Bacterial host strain expressing recombinant DsbC and having reduced Tsp activity

ABSTRACT

The present invention provides a recombinant gram-negative bacterial cell, characterized in that the cell comprises a recombinant polynucleotide encoding DsbC and has reduced Tsp protein activity compared to a wild-type cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2011/050416, filed Jan. 13, 2011.

The invention relates to a recombinant bacterial host strain,particularly E. coli. The invention also relates to a method forproducing a protein of interest in such a cell.

BACKGROUND OF THE INVENTION

Bacterial cells, such as E. coli, are commonly used for producingrecombinant proteins. There are many advantages to using bacterialcells, such as E. coli, for producing recombinant proteins particularlydue to the versatile nature of bacterial cells as host cells allowingthe gene insertion via plasmids. E. coli have been used to produce manyrecombinant proteins including human insulin.

Despite the many advantages to using bacterial cells to producerecombinant proteins, there are still significant limitations includingthe difficulty of producing protease sensitive proteins. Proteases playan important role in turning over old, damaged or miss-folded proteinsin the E. coli periplasm and cytoplasm. Bacterial proteases act todegrade the recombinant protein of interest, thereby often significantlyreducing the yield of active protein.

A number of bacterial proteases have been identified. In E. coliproteases including Protease III (ptr), DegP, OmpT, Tsp, prlC, ptrA,ptrB, pepA-T, tsh, espc, eatA, clpP and Ion have been identified.

Tsp (also known as Pre) is a 60 kDa periplasmic protease. The firstknown substrate of Tsp was Penicillin-binding protein-3 (PBP3)(Determination of the cleavage site involved in C-terminal processing ofpenicillin-binding protein 3 of Escherichia coli; Nagasawa H, SakagamiY, Suzuki A, Suzuki H, Hara H, Hirota Y. J Bacteriol. 1989 November;171(11):5890-3 and Cloning, mapping and characterization of theEscherichia coli Tsp gene which is involved in C-terminal processing ofpenicillin-binding protein 3; Hara H, Yamamoto Y, Higashitani A, SuzukiH, Nishimura Y. J Bacteriol. 1991 August; 173 (15):4799-813) but it waslater discovered that the Tsp was also able to cleave phage tailproteins and, therefore, it was renamed as Tail Specific Protease (Tsp)(Silber et al., Proc. Natl. Acad. Sci. USA, 89: 295-299 (1992)). Silberet al. (Deletion of the prc(tsp) gene provides evidence for additionaltail-specific proteolytic activity in Escherichia coli K-12; Silber, K.R., Sauer, R. T.; Mol Gen Genet 1994 242:237-240) describes a prcdeletion strain (KS 1000) wherein the mutation was created by replacinga segment of the prc gene with a fragment comprising a Kan^(r) marker.

The reduction of Tsp (prc) activity is desirable to reduce theproteolysis of proteins of interest. However, it was found that cellslacking protease prc show thermosensitive growth at low osmolarity. Haraet al isolated thermoresistant revertants containing extragenicsuppressor (spr) mutations (Hara et al., Microbial Drug Resistance, 2:63-72 (1996)). Spr is an 18 kDa membrane bound periplasmic protease andthe substrates of spr are Tsp and peptidoglycans in the outer membraneinvolved in cell wall hydrolysis during cell division. The spr gene isdesignated as UniProtKB/Swiss-Prot P0AFV4 (SPR_ECOLI).

Improved protease deficient strains comprising mutant spr gene have beendescribed. Chen et al (Chen C, Snedecor B, Nishihara J C, Joly J C,McFarland N, Andersen D C, Battersby J E, Champion K M. BiotechnolBioeng. 2004 Mar 5;85(5):463-74) describes the construction of E. colistrains carrying different combinations of mutations in prc (Tsp) andanother protease, DegP, created by amplifying the upstream anddownstream regions of the gene and ligating these together on a vectorcomprising selection markers and a sprW174R mutation (High-levelaccumulation of a recombinant antibody fragment in the periplasm ofEscherichia coli requires a triple-mutant (ΔDegP Δprc sprW174R) hoststrain. The combination of the ΔDegP, Δprc and sprW174R mutations werefound to provide the highest levels of antibody light chain, antibodyheavy chain and F(ab′)2-LZ. EP1341899 discloses an E. coli strain thatis deficient in chromosomal DegP and prc encoding proteases DegP andPre, respectively, and harbors a mutant spr gene that encodes a proteinthat suppresses growth phenotypes exhibited by strains harboring prcmutants.

Protein disulphide isomerase is an enzyme that catalyzes the formationand breakage of disulphide bonds between cysteine residues withinproteins as they fold. It is known to co-express proteins which catalyzethe formation of disulphide bonds to improve protein expression in ahost cell. WO98/56930 discloses a method for producing heterologousdisulfide bond-containing polypeptides in bacterial cells wherein aprokaryotic disulfide isomerase, such as DsbC or DsbG is co-expressedwith a eukaryotic polypeptide. U.S. Pat. No. 6,673,569 discloses anartificial operon comprising polynucleotides encoding each of DsbA,DsbB, DsbC and DsbD for use in producing a foreign protein. EP0786009discloses a process for producing a heterologous polypeptide in bacteriawherein the expression of nucleic acid encoding DsbA or DsbC is inducedprior to the induction of expression of nucleic acid encoding theheterologous polypeptide.

DsbC is a prokaryotic protein found in the periplasm of E. coli whichcatalyzes the formation of disulphide bonds in E. coli. DsbC has anamino acid sequence length of 236 (including signal peptide) and amolecular weight of 25.6 KDa (UniProt No. P0AEG6). DsbC was firstidentified in 1994 (Missiakas et al. The Escherichia coli dsbC (xprA)gene encodes a periplasmic protein involved in disulfide bond formation,The EMBO Journal vol 13, no 8, p 2013-2020, 1994 and Shevchik et al.Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemiand Escherichia coli with disulfide isomerase activity, The EMBO Jounralvol 13, no 8, p 2007-2012, 1994).

It has been surprisingly found that the over-expression of DsbC fromrecombinant DsbC in a gram-negative bacterial cell improves the celllysis phenotype of cells lacking protease Tsp. Accordingly, the presentinventors have provided a new strain having advantageous properties forproducing a protein of interest.

SUMMARY OF THE INVENTION

The present invention provides a recombinant gram-negative bacterialcell, characterized in that the cell:

a) comprises a recombinant polynucleotide encoding DsbC; and

b) has reduced Tsp protein activity compared to a wild-type cell.

In one embodiment the cell comprises a wild-type spr gene. In thisembodiment the cell's genome is preferably isogenic to a wild-typebacterial cell except for the modification required to reduce Tspprotein activity compared to a wild-type cell.

In a further embodiment the cell according to the present invention hasreduced Tsp protein activity compared to a wild-type cell and comprisesa recombinant polynucleotide encoding DsbC and a mutated spr gene. Inthis embodiment the cell's genome is preferably isogenic to a wild-typebacterial cell except for the mutated spr gene and the modificationrequired to reduce Tsp protein activity compared to a wild-type cell.

The gram-negative bacterial cell having the above specific combinationof genetic modifications shows advantageous growth and proteinproduction phenotypes.

The present invention also provides a method for producing a protein ofinterest comprising expressing the protein of interest in a recombinantgram-negative bacterial cell as defined above. The method comprisesculturing a recombinant gram-negative bacterial cell as defined above ina culture medium under conditions effective to express the protein ofinterest and the recombinant polynucleotide encoding DsbC; andrecovering the protein of interest from the periplasm of the recombinantgram-negative bacterial cell and/or the culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth profile of anti-TNF Fab′ expressing strainsMXE001 MXE008 and of anti-TNFα Fab′ and recombinant DsbC expressingstrains MXE001 and MXE008.

FIG. 2 shows anti-TNF Fab′ yield from the periplasm (shaded symbols) andsupernatant (open unshaded symbols) from anti-TNF Fab′ and recombinantDsbC expressing E. coli strains MXE001, MXE008 and MXE009.

FIG. 3 shows anti-TNF Fab′ yield from the periplasm (shaded symbols) andsupernatant (open unshaded symbols) from anti-TNF Fab′ expressing E.coli strains MXE001 and MXE008 and from anti-TNF Fab′ and recombinantDsbC expressing E. coli strains MXE001 and MXE008.

FIG. 4 shows anti-TNF Fab′ yield from the periplasm from Fab A and Fab Bexpressing E. coli strain W3110 and from Fab A and recombinant DsbC orFab B and recombinant DsbC expressing E. coli strain MXE008.

FIG. 5 a shows the 5′ end of the wild type ptr (protease III) andknockout mutated ptr (protease III) protein and gene sequences.

FIG. 5 b shows the 5′ end of the wild type Tsp and knockout mutated Tspprotein and gene sequences.

FIG. 5 c shows a region of the wild type DegP and mutated DegP proteinand gene sequences.

FIG. 6 shows the construction of a vector for use in producing a cellaccording to an embodiment of the present invention.

FIG. 7 shows the comparative growth profiles of 5 L and 200 Lfermentations of anti-TNF Fab′ and recombinant DsbC expressing E. colistrain MXE008.

FIG. 8 shows the comparative Fab′ titres of 5 L and 200 L fermentationsof anti-TNF Fab′ and recombinant DsbC expressing E. coli strain MXE008.

FIG. 9 shows the comparative growth profiles of fermentations ofanti-TNF Fab′ and recombinant DsbC expressing E. coli strains MXE008 andMXE009.

FIG. 10 shows the comparative Fab′ titres of fermentations of anti-TNFFab′ and recombinant DsbC expressing E. coli strain MXE008 and MXE009.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the DNA sequence of the wild-type Tsp gene including the6 nucleotides ATGAAC upstream of the start codon.

SEQ ID NO:2 is the amino acid sequence of the wild-type Tsp protein.

SEQ ID NO:3 is the DNA sequence of a mutated knockout Tsp gene includingthe 6 nucleotides ATGAAT upstream of the start codon.

SEQ ID NO:4 is the DNA sequence of the wild-type Protease III gene.

SEQ ID NO:5 is the amino acid sequence of the wild-type Protease IIIprotein.

SEQ ID NO:6 is the DNA sequence of a mutated knockout Protease III gene.

SEQ ID NO:7 is the DNA sequence of the wild-type DegP gene.

SEQ ID NO:8 is the amino acid sequence of the wild-type DegP protein.

SEQ ID NO:9 is the DNA sequence of a mutated DegP gene.

SEQ ID NO:10 is the amino acid sequence of a mutated DegP protein.

SEQ ID NO: 11 is the amino acid sequence of the light chain variableregion of an anti-TNF antibody.

SEQ ID NO:12 is the amino acid sequence of the heavy chain variableregion of an anti-TNF antibody.

SEQ ID NO:13 is the amino acid sequence of the light chain of ananti-TNF antibody.

SEQ ID NO:14 is the amino acid sequence of the heavy chain of ananti-TNF antibody.

SEQ ID NO: 15 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated Tsp gene comprising the Ase I restriction site.

SEQ ID NO: 16 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated Tsp gene comprising the Ase I restriction site.

SEQ ID NO: 17 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated Protease III gene comprising the Ase I restrictionsite.

SEQ ID NO: 18 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated Protease III gene comprising the Ase I restrictionsite.

SEQ ID NO: 19 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated DegP gene comprising the Ase I restriction site.

SEQ ID NO: 20 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated DegP gene comprising the Ase I restriction site.

SEQ ID NO: 21 is the sequence of the wild-type spr gene including thesignal sequence which is the first 26 amino acid residues. SEQ ID NO:22is the sequence of the wild-type spr gene without the signal sequence.

SEQ ID NO: 23 is the nucleotide sequence of a mutated OmpT sequencecomprising D210A and H212A mutations.

SEQ ID NO: 24 is the amino acid sequence of a mutated OmpT sequencecomprising D210A and H212A mutations.

SEQ ID NO: 25 is the nucleotide sequence of a mutated knockout OmpTsequence.

SEQ ID NO: 26 is the nucleotide sequence of his-tagged DsbC.

SEQ ID NO: 27 is the amino acid sequence of his-tagged DsbC.

SEQ ID NO: 28 shows the amino acid sequence of CDRH1 of hTNF40.

SEQ ID NO: 29 shows the amino acid sequence of CDRH2 of hTNF40 which isa hybrid CDR wherein the C-terminal six amino acids are from the H2 CDRsequence of a human subgroup 3 germline antibody and the amino acidchanges to the sequence resulting from this hybridisation are underlinedas follows: WINTYIGEPI YADSVKG.

SEQ ID NO: 30 shows the amino acid sequence of CDRH3 of hTNF40.

SEQ ID NO: 31 shows the amino acid sequence of CDRL1 of hTNF40.

SEQ ID NO: 32 shows the amino acid sequence of CDRL2 of hTNF40.

SEQ ID NO: 33 shows the amino acid sequence of CDRL3 of hTNF40.

SEQ ID NO: 34 shows the amino acid sequence of CDRH2 of hTNF40.

SEQ ID NO: 35 shows the sequence of the OmpA oligonucleotide adapter.

SEQ ID NO: 36 shows the oligonucleotide cassette encoding intergenicsequence 1 (IGS1) for E. coli Fab expression.

SEQ ID NO: 37 shows the oligonucleotide cassette encoding intergenicsequence 2 (IGS2) for E. coli Fab expression.

SEQ ID NO: 38 shows the oligonucleotide cassette encoding intergenicsequence 3 (IGS3) for E. coli Fab expression.

SEQ ID NO: 39 shows the oligonucleotide cassette encoding intergenicsequence 4 (IGS4) for E. coli Fab expression.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention will now be described in more detail.

The terms “protein” and “polypeptide” are used interchangeably herein,unless the context indicates otherwise. “Peptide” is intended to referto 10 or less amino acids.

The term “polynucleotide” includes a gene, DNA, cDNA, RNA, mRNA etc.unless the context indicates otherwise.

As used herein, the term “comprising” in context of the presentspecification should be interpreted as “including”.

The non-mutated cell or control cell in the context of the presentinvention means a cell of the same type as the recombinant gram-negativecell of the invention wherein the cell has not been modified to reduceTsp protein activity and to carry the recombinant DsbC sequence andoptionally the mutant spr gene. For example, a non-mutated cell may be awild-type cell and may be derived from the same population of host cellsas the cells of the invention before modification to introduce anymutations.

The expressions “cell”, “cell line”, “cell culture” and “strain” areused interchangeably.

The expression “phenotype of a cell comprising a mutated Tsp gene” inthe context of the present invention means the phenotype exhibited by acell harbouring a mutant Tsp gene. Typically cells comprising a mutantTsp gene may lyse, especially at high cell densities. The lysis of thesecells causes any recombinant protein to leak into the supernatant. Cellscarrying mutated Tsp gene may also show thermosensitive growth at lowosmolarity. For example, the cells exhibit no or reduced growth rate orthe cells die in hypotonic media at a high temperature, such as at 40°C. or more.

The term “isogenic” in the context of the present invention means thatthe cell's genome comprises the same or substantially the same nucleicacid sequence(s) compared to the wild-type cell from which the cell isderived except for the modification required to reduce Tsp proteinactivity compared to a wild-type cell and optionally the mutated sprgene. In this embodiment the genome of the cell comprises no furthernon-naturally occurring or genetically engineered mutations. In oneembodiment the genome of the cell of the present invention hassubstantially the same or the same genomic sequence compared towild-type cell except for the modification required to reduce Tspprotein activity compared to a wild-type cell and optionally the mutatedspr gene. In one embodiment the cell according to the present inventionmay have substantially the same genomic sequence compared to thewild-type cell except for the modification required to reduce Tspprotein activity compared to a wild-type cell and optionally the mutatedspr gene taking into account any naturally occurring mutations which mayoccur. In one embodiment, the cell according to the present inventionmay have exactly the same genomic sequence compared to the wild-typecell except for the modification required to reduce Tsp protein activitycompared to a wild-type cell and optionally the mutated spr gene.

The recombinant polynucleotide encoding DsbC may be present on asuitable expression vector transformed into the cell and/or integratedinto the host cell's genome. In the embodiment where the polynucleotideencoding DsbC is inserted into the host's genome, the cell's genome willalso differ from a wild-type cell due to the inserted polynucleotidesequence encoding the DsbC. Preferably the polynucleotide encoding DsbCis in an expression vector in the cell thereby causing minimaldisruption to the host cell's genome.

In one embodiment the cell of the present invention comprises apolynucleotide encoding the protein of interest. In this embodiment, thepolynucleotide encoding the protein of interest may be contained withina suitable expression vector transformed into the cell and/or integratedinto the host cell's genome. In the embodiment where the polynucleotideencoding the protein of interest is inserted into the host's genome, thegenome will also differ from a wild-type cell due to the insertedpolynucleotide sequence encoding the protein of interest. Preferably thepolynucleotide encoding the protein of interest is in an expressionvector in the cell thereby causing minimal disruption to the host cell'sgenome.

The term “wild-type” in the context of the present invention means astrain of a gram-negative bacterial cell as it may occur in nature ormay be isolated from the environment, which does not carry anygenetically engineered mutations. An example of a wild-type strain of E.coli is W3110, such as W3110 K-12 strain.

The present inventors have provided a recombinant gram-negativebacterial cell suitable for expressing a protein of interest which hasreduced Tsp protein activity compared to a wild-type cell and comprisesa recombinant polynucleotide encoding DsbC.

The cells according to the present invention comprise a recombinantpolynucleotide encoding DsbC. As used herein, a “recombinantpolypeptide” refers to a protein that is constructed or produced usingrecombinant DNA technology. The polynucleotide sequence encoding DsbCmay be identical to the endogenous sequence encoding DsbC found inbacterial cells. Alternatively, the recombinant polynucleotide sequenceencoding DsbC is a mutated version of the wild-type DsbC sequence, forexample having a restriction site removed, such as an EcoRI site, and/ora sequence encoding a his-tag. An example modified DsbC nucleotidesequence for use in the present invention is shown in SEQ ID NO: 26,which encodes the his-tagged DsbC amino acid sequence shown in SEQ IDNO: 27.

We have found that the specific combination of the expression ofrecombinant polynucleotide encoding DsbC in a bacterial cell which hasreduced Tsp protein activity compared to a wild-type cell and in apreferred embodiment additionally a mutated spr gene, provides animproved host for expressing proteins of interest. The specificcombination of the above genetic mutations provide new strains whichhave improved cell health and growth phenotype compared to cellscarrying a knockout mutated Tsp gene. Cells carrying mutated Tsp genemay have a good cell growth rate but one limitation of these cells istheir tendency to lyse, especially at high cell densities. Accordinglythe phenotype of a cell comprising a mutated Tsp gene is a tendency tolyse, especially at high cell densities. However, expression of DsbC inthe cells of the present invention, suppresses the reduced Tsp phenotypeand, therefore, the cell exhibits reduced lysis.

The cells according to the present invention exhibit improved proteinproduction yield compared to cells carrying a knockout mutated Tsp gene.The improved protein yield may be the rate of protein production and/orthe duration of protein production from the cell. The improved proteinyield may be the periplasmic protein yield and/or the supernatantprotein yield. In one embodiment the cells of the present invention showimproved periplasmic protein yield compared to a cell carrying a mutatedTsp gene due to reduced leakage from the cell. The recombinant bacterialcells may be capable of faster rate of production of a protein ofinterest and, therefore, the same quantity of a protein of interest maybe produced in a shorter time compared to a cell comprising a mutatedTsp gene. The faster rate of production of a protein of interest may beespecially significant over the initial period of growth of the cell,for example over the first 5, 10, 20 or 30 hours post induction ofprotein expression.

The cells according to the present invention preferably express amaximum yield in the periplasm and/or media of approximately 1.0 g/L,1.5 g/L, 1.8 g/L, 2.0 g/L, 2.4 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L or 4.0 g/Lof a protein of interest.

The cells provided by the present invention have reduced proteaseactivity compared to wild-type cell, which may reduce proteolysis of arecombinant protein of interest, particularly proteins of interest whichare proteolytically sensitive to the Tsp protease. Therefore, the cellsprovided by the present invention provide higher yield of the intactproteins, preferably of the protein of interest and a lower yield, orpreferably no proteolytic fragments of proteins, preferably of theprotein of interest, compared to a wild-type bacterial cell.

The skilled person would easily be able to test a candidate cell cloneto see if it has the desired yield of a protein of interest usingmethods well known in the art including a fermentation method, ELISA andprotein G HPLC. Suitable fermentation methods are described in HumphreysD P, et al. (1997). Formation of dimeric Fabs in E. coli: effect ofhinge size and isotype, presence of interchain disulphide bond, Fab′expression levels, tail piece sequences and growth conditions. J.IMMUNOL. METH 209: 193-202; Backlund E. Reeks D. Markland K. Weir N.Bowering L. Larsson G. Fedbatch design for periplasmic product retentionin Escherichia coli, Journal Article. Research Support, Non-U.S. Gov'tJournal of Biotechnology. 135(4):358-65, 2008 Jul. 31; Champion K M.Nishihara J C. Joly J C. Arnott D. Similarity of the Escherichia coliproteome upon completion of different biopharmaceutical fermentationprocesses. [Journal Article] Proteomics. 1(9):1133-48, 2001 September;and Horn U. Strittmatter W. Krebber A. Knupfer U. Kujau M. Wenderoth R.Muller K. Matzku S. Pluckthun A. Riesenberg D. High volumetric yields offunctional dimeric miniantibodies in Escherichia coli, using anoptimized expression vector and high-cell-density fermentation undernon-limited growth conditions, Journal Article. Research Support,Non-U.S. Gov't Applied Microbiology & Biotechnology. 46(5-6):524-32,1996 December. The skilled person would also easily be able to testsecreted protein to see if the protein is correctly folded using methodswell known in the art, such as protein G HPLC, circular dichroism, NMR,X-Ray crystallography and epitope affinity measurement methods.

In one embodiment the cell according to the present invention alsoexpresses one or more further proteins as follows:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD; and/or    -   one or more protein capable of facilitating protein secretion or        translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and/or    -   one or more proteins capable of facilitating disulphide bond        formation, such as DsbA, DsbB, DsbD, DsbG.

One of more of the above proteins may be integrated into the cell'sgenome and/or inserted in an expression vector.

In one embodiment the cell according to the present invention does notcomprise recombinant polynucleotide encoding one or more of thefollowing further proteins:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD;    -   one or more protein capable of facilitating protein secretion or        translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and    -   one or more proteins capable of facilitating disulphide bond        formation, such as DsbA, DsbB, DsbD, DsbG.

In embodiments of the present invention the cell further comprises amutated spr gene. The spr protein is an E. coli membrane boundperiplasmic protease.

The wild-type amino acid sequence of the spr protein is shown in SEQ IDNO:21 with the signal sequence at the N-terminus and in SEQ ID NO:22without the signal sequence of 26 amino acids (according to UniProtAccession Number P0AFV4). The amino acid numbering of the spr proteinsequence in the present invention includes the signal sequence.Accordingly, the amino acid 1 of the spr protein is the first amino acid(Met) shown in SEQ ID NO: 21.

In the embodiments wherein the cell according to the present inventioncomprises a mutated spr gene, the mutated spr gene is preferably thecell's chromosomal spr gene.

The mutated spr gene encodes a spr protein capable of suppressing thephenotype of a cell comprising a mutated Tsp gene. Cells carrying amutated Tsp gene may have a good cell growth rate but one limitation ofthese cells is their tendency to lyse, especially at high celldensities. Accordingly the phenotype of a cell comprising a mutated Tspgene is a tendency to lyse, especially at high cell densities. Cellscarrying a mutated Tsp gene also show thermosensitive growth at lowosmolarity. However, the spr mutations carried by the cells of thepresent invention, when introduced into a cell having reduced Tspactivity suppress the reduced Tsp phenotype and, therefore, the cellexhibits reduced lysis, particularly at a high cell density. The growthphenotype of a cell may be easily measured by a person skilled in theart during shake flask or high cell density fermentation technique. Thesuppression of the cell lysis phenotype may be been seen from theimproved growth rate and/or recombinant protein production, particularlyin the periplasm, exhibited by a cell carrying spr mutant and havingreduced Tsp activity compared to a cell carrying the Tsp mutant and awild-type spr.

The cells according to the present invention preferably comprise amutant spr gene encoding a spr protein having a mutation at one or moreamino acids selected from N31, R62, I70, Q73, C94, S95, V98, Q99, R100,L108, Y115, D133, V135, L136, G140, R144, H145, G147, H157 and W174,more preferably at one or more amino acids selected from C94, S95, V98,Y115, D133, V135, H145, G147, H157 and W174. Preferably the mutant sprgene encodes a spr protein having a mutation at one or more amino acidsselected from N31, R62, I70, Q73, C94, S95, V98, Q99, R100, L108, Y115,D133, V135, L136, G140, R144, H145, G147 and H157, more preferably atone or more amino acids selected from C94, S95, V98, Y115, D133, V135,H145, G147 and H157. In this embodiment, the spr protein preferably doesnot have any further mutations. Preferably, the mutant spr gene encodesa spr protein having a mutation at one or more amino acids selected fromN31, R62, I70, Q73, S95, V98, Q99, R100, L108, Y115, D133, V135, L136,G140, R144 and G147, more preferably at one or more amino acids selectedfrom S95, V98, Y115, D133, V135 and G147. In this embodiment, the sprprotein preferably does not have any further mutations.

The present inventors have identified spr mutations which are capable ofsuppressing the growth phenotype of a cell comprising a mutated Tspgene.

The inventors have also surprisingly found that cells carrying arecombinant DsbC gene, a new mutated spr gene and having reduced Tspprotein activity compared to a wild-type cell exhibit increased cellgrowth rate and increased cell survival duration compared to a cellcomprising a mutated Tsp gene. Specifically, cells carrying arecombinant DsbC gene, a spr mutation and having reduced Tsp proteinactivity exhibit reduced cell lysis phenotype compared to cells carryinga mutated Tsp gene.

The mutation of one or more of the above spr amino acids may be anysuitable missense mutation to one, two or three of the nucleotidesencoding the amino acid. The mutation changes the amino acid residue toany suitable amino acid which results in a mutated spr protein capableof suppressing the phenotype of a cell comprising a mutated Tsp gene.The missense mutation may change the amino acid to one which is adifferent size and/or has different chemical properties compared to thewild-type amino acid.

In one embodiment the mutation is to one, two or three of the catalytictriad of amino acid residues of C94, H145, and H157 (Solution NMRStructure of the NlpC/P60 Domain of Lipoprotein Spr from Escherichiacoli Structural Evidence for a Novel Cysteine Peptidase Catalytic Triad,Biochemistry, 2008, 47, 9715-9717).

Accordingly, the mutated spr gene may comprise:

-   -   a mutation to C94; or    -   a mutation to H145; or    -   a mutation to H157; or    -   a mutation to C94 and H145; or    -   a mutation to C94 and H157; or    -   a mutation to H145 and H157; or    -   a mutation to C94, H145 and H157.

In this embodiment, the spr protein preferably does not have any furthermutations.

One, two or three of C94, H145 and H157 may be mutated to any suitableamino acid which results in a spr protein capable of suppressing thephenotype of a cell comprising a mutated Tsp gene. For example, one, twoor three of C94, H145, and H157 may be mutated to a small amino acidsuch as Gly or Ala. Accordingly, the spr protein may have one, two orthree of the mutations C94A, H145A and H157A. Preferably, the spr genecomprises the missense mutation H145A, which has been found to produce aspr protein capable of suppressing the phenotype of a cell comprising amutated Tsp gene.

The designation for a substitution mutant herein consists of a letterfollowed by a number followed by a letter. The first letter designatesthe amino acid in the wild-type protein. The number refers to the aminoacid position where the amino acid substitution is being made, and thesecond letter designates the amino acid that is used to replace thewild-type amino acid.

In a preferred embodiment the mutant spr protein comprises a mutation atone or more amino acids selected from N31, R62, I70, Q73, S95, V98, Q99,R100, L108, Y115, D133, V135, L136, G140, R144 and G147, preferably amutation at one or more amino acids selected from S95, V98, Y115, D133,V135 and G147. In this embodiment, the spr protein preferably does nothave any further mutations. Accordingly, the mutated spr gene maycomprise:

-   -   a mutation to N31; or    -   a mutation to R62; or    -   a mutation to 170; or    -   a mutation to Q73; or    -   a mutation to S95; or    -   a mutation to V98; or    -   a mutation to Q99; or    -   a mutation to R100; or    -   a mutation to L108; or    -   a mutation to Y115; or    -   a mutation to D133; or    -   a mutation to V 135; or    -   a mutation to L136; or    -   a mutation to G140; or    -   a mutation to R144; or    -   a mutation to G147.

In one embodiment the mutant spr protein comprises multiple mutations toamino acids:

-   -   S95 and Y115; or    -   N31, Q73, R100 and G140 ; or    -   Q73, R100 and G140; or    -   R100 and G140; or    -   Q73 and G140; or    -   Q73 and R100;or    -   R62, Q99 and R144 ;or    -   Q99 and R144.

One or more of the amino acids N31, R62, I70, Q73, S95, V98, Q99, R100,L108, Y115, D133, V135, L136, G140, R144 and G147 may be mutated to anysuitable amino acid which results in a spr protein capable ofsuppressing the phenotype of a cell comprising a mutated Tsp gene. Forexample, one or more of N31, R62, I70, Q73, S95, V98, Q99, R100, L108,Y115, D133, V135, L136, G140 and R144 may be mutated to a small aminoacid such as Gly or Ala.

In a preferred embodiment the spr protein comprises one or more of thefollowing mutations: N31Y, R62C, 170T, Q73R, S95F, V98E, Q99P, R100G,L108S, Y115F, D133A, V135D or V135G, L136P, G140C, R144C and G147C.Preferably the spr protein comprises one or more of the followingmutations: S95F, V98E, Y115F, D133A, V135D or V135G and G147C. In thisembodiment, the spr protein preferably does not have any furthermutations.

In one embodiment the spr protein has one mutation selected from N31Y,R62C, I70T, Q73R, S95F, V98E, Q99P, R100G, L108S, Y115F, D133A, V135D orV135G, L136P, G140C, R144C and G147C. In this embodiment, the sprprotein preferably does not have any further mutations.

In a further embodiment the spr protein has multiple mutations selectedfrom:

-   -   S95F and Y115F    -   N31Y, Q73R, R100G and G140C;    -   Q73R, R100G and G140C;    -   R100G and G140C;    -   Q73R and G140C;    -   Q73R and R100G;    -   R62C, Q99P and R144C; or    -   Q99P and R144C.

Preferably, the mutant spr gene encodes an spr protein having a mutationselected from C94A, D133A, H145A and H157A.

In a further embodiment the mutated spr gene encodes a spr proteinhaving the mutation W174R. In an alternative embodiment the spr proteindoes not have the mutation W174R.

The cell according to the present invention has reduced Tsp proteinactivity compared to a wild-type cell. The expression “reduced Tspprotein activity compared to a wild-type cell” means that the Tspactivity of the cell is reduced compared to the Tsp activity of awild-type cell. The cell may be modified by any suitable means to reducethe activity of Tsp.

In one embodiment the reduced Tsp activity is from modification of theendogenous polynucleotide encoding Tsp and/or associated regulatoryexpression sequences. The modification may reduce or stop Tsp genetranscription and translation or may provide an expressed Tsp proteinhaving reduced protease activity compared to the wild-type Tsp protein.

In one embodiment an associated regulatory expression sequence ismodified to reduce Tsp expression. For example, the promoter for the Tspgene may be mutated to prevent expression of the gene.

In a preferred embodiment the cell according to the present inventioncarries a mutated Tsp gene encoding a Tsp protein having reducedprotease activity or a knockout mutated Tsp gene. Preferably thechromosomal Tsp gene is mutated.

As used herein, “Tsp gene” means a gene encoding protease Tsp (alsoknown as Prc) which is a periplasmic protease capable of acting onPenicillin-binding protein-3 (PBP3) and phage tail proteins. Thesequence of the wild-type Tsp gene is shown in SEQ ID NO: 1 and thesequence of the wild-type Tsp protein is shown in SEQ ID NO: 2.

Reference to the mutated Tsp gene or mutated Tsp gene encoding Tsp,refers to either a mutated Tsp gene encoding a Tsp protein havingreduced protease activity or a knockout mutated Tsp gene, unlessotherwise indicated.

The expression “mutated Tsp gene encoding a Tsp protein having reducedprotease activity” in the context of the present invention means thatthe mutated Tsp gene does not have the full protease activity comparedto the wild-type non-mutated Tsp gene.

Preferably, the mutated Tsp gene encodes a Tsp protein having 50% orless, 40% or less, 30% or less, 20% or less, 10% or less or 5% or lessof the protease activity of a wild-type non-mutated Tsp protein. Morepreferably, the mutated Tsp gene encodes a Tsp protein having noprotease activity. In this embodiment the cell is not deficient inchromosomal Tsp i.e. the Tsp gene sequence has not been deleted ormutated to prevent expression of any form of Tsp protein.

Any suitable mutation may be introduced into the Tsp gene in order toproduce a protein having reduced protease activity. The proteaseactivity of a Tsp protein expressed from a gram-negative bacterium maybe easily tested by a person skilled in the art by any suitable methodin the art, such as the method described in Keiler et al (Identificationof Active Site Residues of the Tsp Protease* THE JOURNAL OF BIOLOGICALCHEMISTRY Vol. 270, No. 48, Issue of December 1, pp. 28864-28868, 1995Kenneth C. Keiler and Robert T. Sauer) wherein the protease activity ofTsp was tested.

Tsp has been reported in Keiler et al (supra) as having an active sitecomprising residues S430, D441 and K455 and residues G375, G376, E433and T452 are important for maintaining the structure of Tsp. Keiler etal (supra) reports findings that the mutated Tsp genes S430A, D441A,K455A, K455H, K455R, G375A, G376A, E433A and T452A had no detectableprotease activity. It is further reported that the mutated Tsp geneS430C displayed about 5-10% wild-type activity. Accordingly, the Tspmutation to produce a protein having reduced protease activity maycomprise a mutation, such as a missense mutation to one or more ofresidues S430, D441, K455, G375, G376, E433 and T452. Preferably the Tspmutation to produce a protein having reduced protease activity maycomprise a mutation, such as a missense mutation to one, two or allthree of the active site residues S430, D441 and K455.

Accordingly the mutated Tsp gene may comprise:

-   -   a mutation to S430; or    -   a mutation to D441; or    -   a mutation to K455; or    -   a mutation to S430 and D441; or    -   a mutation to S430 and K455; or    -   a mutation to D441 and K455; or    -   a mutation to S430, D441 and K455.

One or more of S430, D441, K455, G375, G376, E433 and T452 may bemutated to any suitable amino acid which results in a protein havingreduced protease activity. Examples of suitable mutations are S430A,S430C, D441A, K455A, K455H, K455R, G375A, G376A, E433A and T452A. Themutated Tsp gene may comprise one, two or three mutations to the activesite residues, for example the gene may comprise:

-   -   S430A or S430C; and/or    -   D441A; and/or    -   K455A or K455H or K455R.

Preferably, the Tsp gene has the point mutation S430A or S430C.

The expression “knockout mutated Tsp gene” in the context of the presentinvention means that the Tsp gene comprises one or more mutations whichprevents expression of the Tsp protein encoded by the wild-type gene toprovide a cell deficient in Tsp protein. The knockout gene may bepartially or completely transcribed but not translated into the encodedprotein. The knockout mutated Tsp gene may be mutated in any suitableway, for example by one or more deletion, insertion, point, missense,nonsense and frameshift mutations, to cause no expression of theprotein. For example, the gene may be knocked out by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence.

In a preferred embodiment the Tsp gene is not mutated by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence. In one embodiment the Tsp gene comprises amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon thereby preventing expression of the Tsp protein. Themutation to the start codon may be a missense mutation of one, two orall three of the nucleotides of the start codon. Alternatively oradditionally the start codon may be mutated by an insertion or deletionframeshift mutation. The Tsp gene comprises two ATG codons at the 5′ endof the coding sequence, one or both of the ATG codons may be mutated bya missense mutation. The Tsp gene may be mutated at the second ATG codon(codon 3) to TCG, as shown in FIG. 5 b. The Tsp gene may alternativelyor additionally comprise one or more stop codons positioned downstreamof the gene start codon and upstream of the gene stop codon. Preferablythe knockout mutated Tsp gene comprises both a missense mutation to thestart codon and one or more inserted stop codons. In a preferredembodiment the Tsp gene is mutated to delete “T” from the fifth codonthereby causing a frameshift resulting in stop codons at codons 11 and16, as shown in FIG. 5 b. In a preferred embodiment the Tsp gene ismutated to insert an Ase I restriction site to create a third in-framestop codon at codon 21, as shown in FIG. 5 b.

In a preferred embodiment the knockout mutated Tsp gene has the DNAsequence of SEQ ID NO: 3, which includes the 6 nucleotides ATGAATupstream of the start codon. The mutations which have been made in theknockout mutated Tsp sequence of SEQ ID NO: 3 are shown in FIG. 5 b. Inone embodiment the mutated Tsp gene has the DNA sequence of nucleotides7 to 2048 of SEQ ID NO:3.

In a preferred embodiment of the present invention the recombinantgram-negative bacterial cell further comprises a mutated DegP geneencoding a DegP protein having chaperone activity and reduced proteaseactivity and/or a mutated ptr gene, wherein the mutated ptr gene encodesa Protease III protein having reduced protease activity or is a knockoutmutated ptr gene and/or a mutated OmpT gene, wherein the mutated OmpTgene encodes an OmpT protein having reduced protease activity or is aknockout mutated OmpT gene.

In one embodiment the present invention provides a recombinantgram-negative bacterial cell comprising

a. a recombinant polynucleotide encoding DsbC;

b. a mutated Tsp gene encoding a Tsp protein having reduced proteaseactivity or a knockout mutated Tsp gene;

c. a mutated DegP gene encoding a DegP protein having chaperone activityand reduced protease activity and/or a mutated OmpT wherein the mutatedOmpT gene encodes an OmpT protein having reduced protease activity or isa knockout mutated OmpT gene; and

d. optionally a mutated spr gene.

Preferably in this embodiment the cell's genome is isogenic to awild-type bacterial cell except for the above mutations b, c and d.

In one embodiment the present invention provides a recombinantgram-negative bacterial cell comprising:

a. a recombinant polynucleotide encoding DsbC;

b. a mutated Tsp gene encoding a Tsp protein having reduced proteaseactivity or a knockout mutated Tsp gene;

c. a mutated ptr gene, wherein the mutated ptr gene encodes a ProteaseIII protein having reduced protease activity or is a knockout mutatedptr gene and/or a mutated OmpT wherein the mutated OmpT gene encodes anOmpT protein having reduced protease activity or is a knockout mutatedOmpT gene; and

d. optionally a mutated spr gene.

Preferably in this embodiment the cell's genome is isogenic to awild-type bacterial cell except for the above mutations b, c and d.

In one embodiment the present invention provides a cell comprising

a. a recombinant polynucleotide encoding DsbC;

b. a mutated Tsp gene encoding a Tsp protein having reduced proteaseactivity or a knockout mutated Tsp gene;

c. a mutated DegP gene encoding a DegP protein having chaperone activityand reduced protease activity;

d. a mutated ptr gene, wherein the mutated ptr gene encodes a ProteaseIII protein having reduced protease activity or is a knockout mutatedptr gene;

e. optionally a mutated OmpT wherein the mutated OmpT gene encodes anOmpT protein having reduced protease activity or is a knockout mutatedOmpT gene; and

f. optionally a mutated spr gene.

Preferably in this embodiment the cell's genome is isogenic to awild-type bacterial cell except for the above mutations b, c, d, e andf.

In embodiments of the present invention the cell comprises a mutatedDegP gene. As used herein, “DegP” means a gene encoding DegP protein(also known as HtrA), which has dual function as a chaperone and aprotease (Families of serine peptidases; Rawlings N D, Barrett A J.Methods Enzymol. 1994;244:19-61). The sequence of the non-mutated DegPgene is shown in SEQ ID NO: 7 and the sequence of the non-mutated DegPprotein is shown in SEQ ID NO: 8.

At low temperatures DegP functions as a chaperone and at hightemperatures DegP has a preference to function as a protease (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M) and The proteolytic activity of the HtrA(DegP) protein from Escherichia coli at low temperatures, Skorko-GlonekJ et al Microbiology 2008, 154, 3649-3658).

In the embodiments where the cell comprises the DegP mutation the DegPmutation in the cell provides a mutated DegP gene encoding a DegPprotein having chaperone activity but not full protease activity.

The expression “having chaperone activity” in the context of the presentinvention means that the mutated DegP protein has the same orsubstantially the same chaperone activity compared to the wild-typenon-mutated DegP protein. Preferably, the mutated DegP gene encodes aDegP protein having 50% or more, 60% or more, 70% or more, 80% or more,90% or more or 95% or more of the chaperone activity of a wild-typenon-mutated DegP protein. More preferably, the mutated DegP gene encodesa DegP protein having the same chaperone activity compared to wild-typeDegP.

The expression “having reduced protease activity” in the context of thepresent invention means that the mutated DegP protein does not have thefull protease activity compared to the wild-type non-mutated DegPprotein. Preferably, the mutated DegP gene encodes a DegP protein having50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% orless of the protease activity of a wild-type non-mutated DegP protein.More preferably, the mutated DegP gene encodes a DegP protein having noprotease activity. The cell is not deficient in chromosomal DegP i.e.the DegP gene sequences has not been deleted or mutated to preventexpression of any form of DegP protein.

Any suitable mutation may be introduced into the DegP gene in order toproduce a protein having chaperone activity and reduced proteaseactivity. The protease and chaperone activity of a DegP proteinexpressed from a gram-negative bacterium may be easily tested by aperson skilled in the art by any suitable method such as the methoddescribed in Spiess et al wherein the protease and chaperone activitiesof DegP were tested on MalS, a natural substrate of DegP (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M) and also the method described in Theproteolytic activity of the HtrA (DegP) protein from Escherichia coli atlow temperatures, Skorko-Glonek J et al Microbiology 2008, 154,3649-3658.

DegP is a serine protease and has an active center consisting of acatalytic triad of amino acid residues of His105, Asp135 and Ser210(Families of serine peptidases, Methods Enzymol., 1994, 244:19-61Rawlings N and Barrett A). The DegP mutation to produce a protein havingchaperone activity and reduced protease activity may comprise amutation, such as a missense mutation to one, two or three of His105,Asp135 and Ser210.

Accordingly, the mutated DegP gene may comprise:

-   -   a mutation to His105; or    -   a mutation to Asp135; or    -   a mutation to Ser210; or    -   a mutation to His105 and Asp135; or    -   a mutation to His105 and Ser210; or    -   a mutation to Asp135 and Ser210; or    -   a mutation to His105, Asp135 and Ser210.

One, two or three of His105, Asp135 and Ser210 may be mutated to anysuitable amino acid which results in a protein having chaperone activityand reduced protease activity. For example, one, two or three of His105,Asp135 and Ser210 may be mutated to a small amino acid such as Gly orAla. A further suitable mutation is to change one, two or three ofHis105, Asp135 and Ser210 to an amino acid having opposite propertiessuch as Asp135 being mutated to Lys or Arg, polar His105 being mutatedto a non-polar amino acid such as Gly, Ala, Val or Leu and smallhydrophilic Ser210 being mutated to a large or hydrophobic residue suchas Val, Leu, Phe or Tyr. Preferably, the DegP gene comprises the pointmutation S210A, as shown in FIG. 5 c, which has been found to produce aprotein having chaperone activity but not protease activity (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M).

DegP has two PDZ domains, PDZ1 (residues 260-358) and PDZ2 (residues359-448), which mediate protein-protein interaction (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M). In one embodiment of the present inventionthe degP gene is mutated to delete PDZ1 domain and/or PDZ2 domain. Thedeletion of PDZ1 and PDZ2 results in complete loss of protease activityof the DegP protein and lowered chaperone activity compared to wild-typeDegP protein whilst deletion of either PDZ1 or PDZ2 results in 5%protease activity and similar chaperone activity compared to wild-typeDegP protein (A Temperature-Dependent Switch from Chaperone to Proteasein a Widely Conserved Heat Shock Protein. Cell, Volume 97, Issue 3,Pages 339-347. Spiess C, Beil A, Ehrmann M).

The mutated DegP gene may also comprise a silent non-naturally occurringrestriction site, such as Ase I in order to aid in identification andscreening methods, for example as shown in FIG. 5 c.

The preferred sequence of the mutated DegP gene comprising the pointmutation S210A and an Ase I restriction marker site is provided in SEQID NO: 9 and the encoded protein sequence is shown in SEQ ID NO: 10. Themutations which have been made in the mutated DegP sequence of SEQ IDNO: 9 are shown in FIG. 5 c.

In the embodiments of the present invention wherein the cell comprises amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, one or more of the cells provided by thepresent invention may provide improved yield of correctly foldedproteins from the cell relative to mutated cells wherein the DegP genehas been mutated to knockout DegP preventing DegP expression, such aschromosomal deficient DegP. In a cell comprising a knockout mutated DegPgene preventing DegP expression, the chaperone activity of DegP is lostcompletely whereas in the cell according to the present invention thechaperone activity of DegP is retained whilst the full protease activityis lost. In these embodiments, one or more cells according to thepresent invention have a lower protease activity to prevent proteolysisof the protein whilst maintaining the chaperone activity to allowcorrect folding and transportation of the protein in the host cell.

The skilled person would easily be able to test secreted protein to seeif the protein is correctly folded using methods well known in the art,such as protein G HPLC, circular dichroism, NMR, X-Ray crystallographyand epitope affinity measurement methods.

In these embodiments, one or more cells according to the presentinvention may have improved cell growth compared to cells carrying amutated knockout DegP gene preventing DegP expression. Without wishingto be bound by theory improved cell growth maybe exhibited due to theDegP protease retaining chaperone activity which may increase capacityof the cell to process all proteins which require chaperone activity.Accordingly, the production of correctly folded proteins necessary forthe cell's growth and reproduction may be increased in one or more ofthe cells of the present invention compared to cells carrying a DegPknockout mutation thereby improving the cellular pathways regulatinggrowth. Further, known DegP protease deficient strains are generallytemperature-sensitive and do not typically grow at temperatures higherthan about 28° C. However, the cells according to the present inventionare not temperature-sensitive and may be grown at temperatures of 28° C.or higher, including temperatures of approximately 30° C. toapproximately 37° C., which are typically used for industrial scaleproduction of proteins from bacteria.

In embodiments of the present invention the cell comprises a mutated ptrgene. As used herein, “ptr gene” means a gene encoding Protease III, aprotease which degrades high molecular weight proteins. The sequence ofthe non-mutated ptr gene is shown in SEQ ID NO: 4 and the sequence ofthe non-mutated Protease III protein is shown in SEQ ID NO: 5.

Reference to the mutated ptr gene or mutated ptr gene encoding ProteaseIII, refers to either a mutated ptr gene encoding a Protease III proteinhaving reduced protease activity or a knockout mutated ptr gene, unlessotherwise indicated.

The expression “mutated ptr gene encoding a Protease III protein havingreduced protease activity” in the context of the present invention meansthat the mutated ptr gene does not have the full protease activitycompared to the wild-type non-mutated ptr gene.

Preferably, the mutated ptr gene encodes a Protease III having 50% orless, 40% or less, 30% or less, 20% or less, 10% or less or 5% or lessof the protease activity of a wild-type non-mutated Protease IIIprotein. More preferably, the mutated ptr gene encodes a Protease IIIprotein having no protease activity. In this embodiment the cell is notdeficient in chromosomal ptr i.e. the ptr gene sequence has not beendeleted or mutated to prevent expression of any form of Protease IIIprotein.

Any suitable mutation may be introduced into the ptr gene in order toproduce a Protease III protein having reduced protease activity. Theprotease activity of a Protease III protein expressed from agram-negative bacterium may be easily tested by a person skilled in theart by any suitable method in the art.

The expression “knockout mutated ptr gene” in the context of the presentinvention means that the gene comprises one or more mutations therebycausing no expression of the protein encoded by the gene to provide acell deficient in the protein encoded by the knockout mutated gene. Theknockout gene may be partially or completely transcribed but nottranslated into the encoded protein. The knockout mutated ptr gene maybe mutated in any suitable way, for example by one or more deletion,insertion, point, missense, nonsense and frameshift mutations, to causeno expression of the protein. For example, the gene may be knocked outby insertion of a foreign DNA sequence, such as an antibiotic resistancemarker, into the gene coding sequence.

In a preferred embodiment the gene is not mutated by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence. Preferably the Protease III gene comprise amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon thereby preventing expression of the Protease III protein.

A mutation to the target knockout gene start codon causes loss offunction of the start codon and thereby ensures that the target genedoes not comprise a suitable start codon at the start of the codingsequence. The mutation to the start codon may be a missense mutation ofone, two or all three of the nucleotides of the start codon.

Alternatively or additionally the start codon may be mutated by aninsertion or deletion frameshift mutation.

In a preferred embodiment the ptr gene is mutated to change the ATGstart codon to ATT, as shown in FIG. 5 a.

The knockout mutated ptr gene may alternatively or additionally compriseone or more stop codons positioned downstream of the gene start codonand upstream of the gene stop codon. Preferably the knockout mutated ptrgene comprises both a missense mutation to the start codon and one ormore inserted stop codons.

The one or more inserted stop codons are preferably in-frame stopcodons. However the one or more inserted stop codons may alternativelyor additionally be out-of-frame stop codons. One or more out-of-framestop codons may be required to stop translation where an out-of-framestart codon is changed to an in-frame start codon by an insertion ordeletion frameshift mutation. The one or more stop codons may beintroduced by any suitable mutation including a nonsense point mutationand a frameshift mutation. The one or more stop codons are preferablyintroduced by a frameshift mutation and/or an insertion mutation,preferably by replacement of a segment of the gene sequence with asequence comprising a stop codon. For example an Ase I restriction sitemay be inserted, which comprises the stop codon TAA.

In a preferred embodiment the ptr gene is mutated to insert an in-framestop codon by insertion of an Ase I restriction site, as shown in FIG. 5a. In a preferred embodiment the knockout mutated ptr gene has the DNAsequence of SEQ ID NO: 6. The mutations which have been made in theknockout mutated ptr gene sequence of SEQ ID NO: 6 are shown in FIG. 5a.

The above described knockout mutations are advantageous because theycause minimal or no disruption to the chromosomal DNA upstream ordownstream of the target knockout gene site and do not require theinsertion and retention of foreign DNA, such as antibiotic resistancemarkers, which may affect the cell's suitability for expressing aprotein of interest, particularly therapeutic proteins. Accordingly, oneor more of the cells according to the present invention may exhibitimproved growth characteristics and/or protein expression compared tocells wherein the protease gene has been knocked out by insertion offoreign DNA into the gene coding sequence.

In embodiments of the present invention the cell carries a mutated OmpTgene. As used herein, “OmpT gene” means a gene encoding protease OmpT(outer membrane protease T) which is an outer membrane protease. Thesequence of the wild-type non-mutated OmpT gene is SWISS-PROT P09169.

Reference to a mutated OmpT gene or mutated OmpT gene encoding OmpT,refers to either a mutated OmpT gene encoding a OmpT protein havingreduced protease activity or a knockout mutated OmpT gene, unlessotherwise indicated.

The expression “mutated OmpT gene encoding a OmpT protein having reducedprotease activity” in the context of the present invention means thatthe mutated OmpT gene does not have the full protease activity comparedto the wild-type non-mutated OmpT gene. The mutated OmpT gene may encodea OmpT protein having 50% or less, 40% or less, 30% or less, 20% orless, 10% or less or 5% or less of the protease activity of a wild-typenon-mutated OmpT protein. The mutated OmpT gene may encode a OmpTprotein having no protease activity. In this embodiment the cell is notdeficient in chromosomal OmpT i.e. the OmpT gene sequence has not beendeleted or mutated to prevent expression of any form of OmpT protein.

Any suitable mutation may be introduced into the OmpT gene in order toproduce a protein having reduced protease activity. The proteaseactivity of a OmpT protein expressed from a gram-negative bacterium maybe easily tested by a person skilled in the art by any suitable methodin the art, such as the method described in Kramer et al (Identificationof essential acidic residues of outer membrane protease OmpT supports anovel active site, FEBS Letters 505 (2001) 426-430) and Dekker et al(Substrate Specificity of the Integral Membrane Protease OmpT Determinedby Spatially Addressed Peptide Libraries, Biochemistry 2001, 40,1694-1701).

OmpT has been reported in Kramer et al (Identification of active siteserine and histidine residues in Escherichia coli outer membraneprotease OmpT FEBS Letters 2000 468, 220-224) discloses thatsubstitution of serines, histidines and acidic residues by alaninesresults in ˜10-fold reduced activity for Glu27, Asp97, Asp208 or His101,˜500-fold reduced activity for Ser99 and ˜10000-fold reduced activityfor Asp83, Asp85, Asp210 or His212. Vandeputte-Rutten et al (CrystalStructure of the Outer Membrane Protease OmpT from Escherichia colisuggests a novel catalytic site, The EMBO Journal 2001, Vol 20 No 185033-5039) as having an active site comprising a Asp83-Asp85 pair and aHis212-Asp210 pair. Further Kramer et al (Lipopolysaccharide regionsinvolved in the activation of Escherichia coli outer membrane proteaseOmpT, Eur. J. Biochem. FEBS 2002, 269, 1746-1752) discloses thatmutations D208A, D210A, H212A, H212N, H212Q, G216K, K217G, K217T andR218L in loop L4 all resulted in partial or virtually complete loss ofenzymatic activity.

Accordingly, the OmpT mutation to produce a protein having reducedprotease activity may comprise a mutation, such as a missense mutationto one or more of residues E27, D43, D83, D85, D97, S99, H101 E111,E136, E193, D206, D208, D210, H212 G216, K217, R218 & E250.

One or more of E27, D43, D83, D85, D97, S99, H101 E111, E136, E193,D206, D208, D210, H212 G216, K217, R218 & E250 may be mutated to anysuitable amino acid which results in a protein having reduced proteaseactivity. For example, one of more of E27, D43, D83, D85, D97, S99, H101E111, E136, E193, D206, D208, D210, H212 G216, K217, R218 & E250 may bemutated to alanine. Examples of suitable mutations are E27A, D43A, D83A,D85A, D97A, S99A, H101A E111A, E136A, E193A, D206A, D208A, D210A, H212A,H212N, H212Q, G216K, K217G, K217T, R218L & E250A. In one embodiment themutated OmpT gene comprises D210A and H212A mutations. A suitablemutated OmpT sequence comprising D210A and H212A mutations is shown inSEQ ID NO: 23.

The expression “knockout mutated OmpT gene” in the context of thepresent invention means that the gene comprises one or more mutationsthereby causing no expression of the protein encoded by the gene toprovide a cell deficient in the protein encoded by the knockout mutatedgene. The knockout gene may be partially or completely transcribed butnot translated into the encoded protein. The knockout mutated OmpT genemay be mutated in any suitable way, for example by one or more deletion,insertion, point, missense, nonsense and frameshift mutations, to causeno expression of the protein. For example, the gene may be knocked outby insertion of a foreign DNA sequence, such as an antibiotic resistancemarker, into the gene coding sequence.

In one embodiment the OmpT gene comprises a mutation to the gene startcodon and/or one or more stop codons positioned downstream of the genestart codon and upstream of the gene stop codon thereby preventingexpression of the OmpT protein. The mutation to the start codon may be amissense mutation of one, two or all three of the nucleotides of thestart codon. Alternatively or additionally the start codon may bemutated by an insertion or deletion frameshift mutation.

A suitable mutated knockout OmpT sequence is shown in SEQ ID NO: 24.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ompT gene, such asbeing deficient in chromosomal ompT.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated degP gene, such asbeing deficient in chromosomal degP. In one embodiment the gram-negativebacterial cell according to the present invention does not carry amutated degP gene.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ptr gene, such asbeing deficient in chromosomal ptr.

Many genetically engineered mutations including knockout mutationsinvolve the use of antibiotic resistance markers which allow theselection and identification of successfully mutated cells. However, asdiscussed above, there are a number of disadvantages to using antibioticresistance markers.

A further embodiment of the present invention overcomes the abovedisadvantages of using antibiotic resistance markers wherein the mutatedTsp gene, optionally the mutated spr, optionally the mutated DegP geneand/or a mutated ptr gene and/or a mutated OmpT gene, are mutated tocomprise one or more restriction marker sites. The restriction sites aregenetically engineered into the gene and are non-naturally occurring.The restriction marker sites are advantageous because they allowscreening and identification of correctly modified cells which comprisethe required chromosomal mutations. Cells which have been modified tocarry one or more of the mutated protease genes may be analyzed by PCRof genomic DNA from cell lysates using oligonucleotide pairs designed toamplify a region of the genomic DNA comprising a non-naturally occurringrestriction marker site. The amplified DNA may then be analyzed byagarose gel electrophoresis before and after incubation with a suitablerestriction enzyme capable of digesting the DNA at the non-naturallyoccurring restriction marker site. The presence of DNA fragments afterincubation with the restriction enzyme confirms that the cells have beensuccessfully modified to carry the one or more mutated genes.

In the embodiment wherein the cell carries a knockout mutated ptr genehaving the DNA sequence of SEQ ID NO: 6, the oligonucleotide primersequences shown in SEQ ID NO: 17 and SEQ ID NO:18 may be used to amplifythe region of the DNA comprising the non-naturally occurring Ase Irestriction site from the genomic DNA of transformed cells. Theamplified genomic DNA may then be incubated with Ase I restrictionenzyme and analyzed by gel electrophoresis to confirm the presence ofthe mutated ptr gene in the genomic DNA.

In the embodiment wherein the cell comprises a knockout mutated Tsp genehaving the DNA sequence of SEQ ID NO: 3 or nucleotides 7 to 2048 of SEQID NO:3, the oligonucleotide primer sequences shown in SEQ ID NO: 15 andSEQ ID NO:16 may be used to amplify the region of the DNA comprising thenon-naturally occurring Ase I restriction site from the genomic DNA oftransformed cells. The amplified genomic DNA may then be incubated withAse I restriction enzyme and analyzed by gel electrophoresis to confirmthe presence of the mutated Tsp gene in the genomic DNA.

In the embodiment wherein the cell comprises a mutated DegP gene havingthe DNA sequence of SEQ ID NO: 9, the oligonucleotide primer sequencesshown in SEQ ID NO: 19 and SEQ ID NO:20 may be used to amplify theregion of the DNA comprising the non-naturally occurring Ase Irestriction site from the genomic DNA of transformed cells. Theamplified genomic DNA may then be incubated with Ase I restrictionenzyme and analyzed by gel electrophoresis to confirm the presence ofthe mutated DegP gene in the genomic DNA.

The one or more restriction sites may be introduced by any suitablemutation including by one or more deletion, insertion, point, missense,nonsense and frameshift mutations. A restriction site may be introducedby the mutation of the start codon and/or mutation to introduce the oneor more stop codons, as described above. This embodiment is advantageousbecause the restriction marker site is a direct and unique marker of theknockout mutations introduced.

A restriction maker site may be inserted which comprises an in-framestop codon, such as an Ase I restriction site. This is particularlyadvantageous because the inserted restriction site serves as both arestriction marker site and a stop codon to prevent full transcriptionof the gene coding sequence. For example, in the embodiment wherein astop codon is introduced to the ptr gene by introduction of an Ase Isite, this also creates a restriction site, as shown in FIG. 5 a. Forexample, in the embodiment wherein a stop codon is introduced to the Tspgene at codon 21 by introduction of an Ase I site, this also creates arestriction site, as shown in FIG. 5 b.

A restriction marker site may be inserted by the mutation to the startcodon and optionally one or more further point mutations. In thisembodiment the restriction marker site is preferably an EcoR Irestriction site. This is particularly advantageous because the mutationto the start codon also creates a restriction marker site. For example,in the embodiment wherein the start codon of the ptr gene is changed toATT, this creates an EcoR I marker site, as shown in FIG. 5 a. Forexample, in the embodiment wherein the start codon (codon 3) of the Tspgene is changed from ATG to TCG, as shown in FIG. 1 b, a further pointmutation of codon 2 from AAC to AAT and mutation of codon 3 ATG to TCGcreates an EcoR I restriction marker site, as shown in FIG. 5 b.

In the embodiment of the present invention wherein the cell carries amutated OmpT gene, the one or more restriction sites may be introducedby any suitable mutation including by one or more deletion, insertion,point, missense, nonsense and frameshift mutations. For example, in theembodiment wherein the OmpT gene comprises the mutations D210A andH212A, these mutations introduce silent HindIII restriction site whichmay be used as a selection marker.

In the DegP gene or the spr gene, a marker restriction site may beintroduced using silent codon changes. For example, an Ase I site may beused as a silent restriction marker site, wherein the TAA stop codon isout-of-frame, as shown in FIG. 5 c for the mutated DegP gene.

In the embodiments of the present invention, wherein the ptr gene and/orthe Tsp gene are mutated to encode a Protease III or Tsp having reducedprotease activity, one or more marker restriction site may be introducedusing silent codon changes.

Any suitable gram-negative bacterium may be used as the parental cellfor producing the recombinant cell of the present invention. Suitablegram-negative bacterium include Salmonella typhimurium, Pseudomonasfluorescens, Erwinia carotovora, Shigella, Klebsiella pneumoniae,Legionella pneumophila, Pseudomonas aeruginosa, Acinetobacter baumanniiand E. coli. Preferably the parental cell is E. coli. Any suitablestrain of E. coli may be used in the present invention but preferably awild-type W3110 strain, such as K-12 W3110, is used.

A drawback associated with the protease deficient bacterial strainspreviously created and used to express recombinant proteins is that theycomprise additional mutations to genes involved in cell metabolism andDNA replication such as, for example phoA, fhuA, lac, rec, gal, ara,arg, thi and pro in E. coli strains. These mutations may have manydeleterious effects on the host cell including effects on cell growth,stability, recombinant protein expression yield and toxicity. Strainshaving one or more of these genomic mutations, particularly strainshaving a high number of these mutations, may exhibit a loss of fitnesswhich reduces bacterial growth rate to a level which is not suitable forindustrial protein production. Further, any of the above genomicmutations may affect other genes in cis and/or in trans in unpredictableharmful ways thereby altering the strain's phenotype, fitness andprotein profile. Further, the use of heavily mutated cells is notgenerally suitable for producing recombinant proteins for commercialuse, particularly therapeutics, because such strains generally havedefective metabolic pathways and hence may grow poorly or not at all inminimal or chemically defined media.

In a preferred embodiment, the cells carry only the minimal mutations tointroduce the recombinant polynucleotide encoding DsbC; the modificationrequired to reduce Tsp protein activity and optionally the mutated sprgene. Only minimal mutations are made to the cell's genome to introducerecombinant polynucleotide and mutations. The cells do not carry anyother mutations which may have deleterious effects on the cell's growthand/or ability to express a protein of interest. Accordingly, one ormore of the recombinant host cells of the present invention may exhibitimproved protein expression and/or improved growth characteristicscompared to cells comprising further genetically engineered mutations tothe genomic sequence. The cells provided by the present invention arealso more suitable for use in the production of therapeutic proteinscompared to cells comprising further disruptions to the cell genome.

Accordingly, the present invention also provides a gram-negativebacterial cell which has a genome isogenic to a wild-type bacterial cellexcept for the recombinant the modification required to reduce Tspprotein activity and optionally the mutated spr gene. The cell furthercarries a recombinant polynucleotide encoding DsbC and optionally apolynucleotide sequence encoding a protein of interest.

In a preferred embodiment, the cell is isogenic to a wild-type E. colicell, such as strain W3110, except for the modification required toreduce Tsp protein activity compared to a wild-type cell and optionallythe mutated spr gene. The cell further carries a recombinantpolynucleotide encoding DsbC and optionally a polynucleotide sequenceencoding a protein of interest.

The cell according to the present invention may further comprise apolynucleotide sequence encoding a protein of interest. Thepolynucleotide sequence encoding the protein of interest may beexogenous or endogenous. The polynucleotide sequence encoding theprotein of interest may be integrated into the host's chromosome or maybe non-integrated in a vector, typically a plasmid. Preferably thepolynucleotide is in an expression vector in the cell thereby causingminimal disruption to the host cell's genome.

“Protein of interest” in the context of the present specification isintended to refer to polypeptide for expression, usually a recombinantpolypeptide. However, the protein of interest may be an endogenousprotein expressed from an endogenous gene in the host cell.

As used herein, a “recombinant polypeptide” refers to a protein that isconstructed or produced using recombinant DNA technology. The protein ofinterest may be an exogenous sequence identical to an endogenous proteinor a mutated version thereof, for example with attenuated biologicalactivity, or fragment thereof, expressed from an exogenous vector.Alternatively, the protein of interest may be a heterologous protein,not normally expressed by the host cell.

The protein of interest may be any suitable protein including atherapeutic, prophylactic or diagnostic protein.

In one embodiment the protein of interest is useful in the treatment ofdiseases or disorders including inflammatory diseases and disorders,immune disease and disorders, fibrotic disorders and cancers.

The term “inflammatory disease” or “disorder” and “immune disease ordisorder” includes rheumatoid arthritis, psoriatic arthritis, still'sdisease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerativecolitis, SLE (Systemic Lupus Erythematosus), asthma, allergic rhinitis,atopic dermatitis, multiple sclerosis, vasculitis, Type I diabetesmellitus, transplantation and graft-versus-host disease.

The term “fibrotic disorder” includes idiopathic pulmonary fibrosis(IPF), systemic sclerosis (or scleroderma), kidney fibrosis, diabeticnephropathy, IgA nephropathy, hypertension, end-stage renal disease,peritoneal fibrosis (continuous ambulatory peritoneal dialysis), livercirrhosis, age-related macular degeneration (ARMD), retinopathy, cardiacreactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty,coronary bypass surgery, arthroplasty and cataract surgery.

The term “cancer” includes a malignant new growth that arises fromepithelium, found in skin or, more commonly, the lining of body organs,for example: breast, ovary, prostate, lung, kidney, pancreas, stomach,bladder or bowel. Cancers tend to infiltrate into adjacent tissue andspread (metastasise) to distant organs, for example: to bone, liver,lung or the brain.

The protein may be a proteolytically-sensitive polypeptide, i.e.proteins that are prone to be cleaved, susceptible to cleavage, orcleaved by one or more gram-negative bacterial, such as E. coli,proteases, either in the native state or during secretion. In oneembodiment the protein of interest is proteolytically-sensitive to aprotease selected from DegP, Protease III and Tsp. In one embodiment theprotein of interest is proteolytically-sensitive to the protease Tsp. Inone embodiment the protein of interest is proteolytically-sensitive tothe proteases DegP and Protease III. In one embodiment the protein ofinterest is proteolytically sensitive to the proteases DegP and Tsp. Inone embodiment the protein of interest is proteolytically-sensitive tothe proteases Tsp and Protease III. In one embodiment the protein ofinterest is proteolytically sensitive to the proteases DegP, ProteaseIII and Tsp.

Preferably the protein is a eukaryotic polypeptide.

The protein of interest expressed by the cells according to theinvention may, for example be an immunogen, a fusion protein comprisingtwo heterologous proteins or an antibody. Antibodies for use as theprotein of interest include monoclonal, multi-valent, multi-specific,humanized, fully human or chimeric antibodies. The antibody can be fromany species but is preferably derived from a monoclonal antibody, ahuman antibody, or a humanized fragment. The antibody can be derivedfrom any class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass ofimmunoglobulin molecule and may be obtained from any species includingfor example mouse, rat, shark, rabbit, pig, hamster, camel, llama, goator human. Parts of the antibody fragment may be obtained from more thanone species for example the antibody fragments may be chimeric. In oneexample the constant regions are from one species and the variableregions from another.

The antibody may be a complete antibody molecule having full lengthheavy and light chains or a fragment thereof, e.g. VH, VL, VHH, Fab,modified Fab, Fab′, F(ab′)₂, Fv, scFv fragment, Fab-Fv, or a dualspecificity antibody, such as a Fab-dAb, as described inPCT/GB2008/003331.

The antibody may be specific for any target antigen. The antigen may bea cell-associated protein, for example a cell surface protein on cellssuch as bacterial cells, yeast cells, T-cells, endothelial cells ortumour cells, or it may be a soluble protein. Antigens of interest mayalso be any medically relevant protein such as those proteinsupregulated during disease or infection, for example receptors and/ortheir corresponding ligands. Particular examples of cell surfaceproteins include adhesion molecules, for example integrins such as β1integrins e.g. VLA-4, E-selectin, P selectin or L-selectin, CD2, CD3,CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33,CD38, CD40, CD40L, CD45, CDW52, CD69, CD134 (OX40), ICOS, BCMP7, CD137,CD27L, CDCP1, CSF1 or CSF1-Receptor, DPCR1, DPCR1, dudulin2, FLJ20584,FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK,MAL2, MRP2, nectin-like2, NKCC1, PTK7, RAIG1, TCAM1, SC6, BCMP101,BCMP84, BCMP11, DTD, carcinoembryonic antigen (CEA), human milk fatglobulin (HMFG1 and 2), MHC Class I and MHC Class II antigens, KDR andVEGF, and where appropriate, receptors thereof

Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, IL-12, IL-13, IL-14, IL-16 or IL-17, such as IL17Aand/or IL17F, viral antigens for example respiratory syncytial virus orcytomegalovirus antigens, immunoglobulins, such as IgE, interferons suchas interferon α, interferon β or interferon γ, tumour necrosis factorTNF (formerly known as tumour necrosis factor-α), tumor necrosisfactor-β, colony stimulating factors such as G-CSF or GM-CSF, andplatelet derived growth factors such as PDGF-α, and PDGF-β and whereappropriate receptors thereof Other antigens include bacterial cellsurface antigens, bacterial toxins, viruses such as influenza, EBV,HepA, B and C, bioterrorism agents, radionuclides and heavy metals, andsnake and spider venoms and toxins.

In one embodiment, the antibody may be used to functionally alter theactivity of the antigen of interest. For example, the antibody mayneutralize, antagonize or agonise the activity of said antigen, directlyor indirectly.

The present invention also provides a recombinant gram-negativebacterial cell comprising a recombinant polynucleotide encoding DsbC, amutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp proteinhaving reduced protease activity or is a knockout mutated Tsp gene, anda polynucleotide sequence encoding an antibody or an antigen bindingfragment thereof specific for TNF. In a preferred embodiment the cellfurther comprises a mutant spr gene encoding a mutant spr protein.

In a preferred embodiment the protein of interest expressed by the cellsaccording to the present invention is an anti-TNF antibody, morepreferably an anti-TNF Fab′, as described in WO01/094585 (the contentsof which are incorporated herein by reference).

In a one embodiment the antibody having specificity for human TNFα,comprises a heavy chain wherein the variable domain comprises a CDRhaving the sequence shown in SEQ ID NO:28 for CDRH1, the sequence shownin SEQ ID NO:29 or SEQ ID NO:34 for CDRH2 or the sequence shown in SEQID NO:30 for CDRH3.

In one embodiment the antibody comprises a light chain wherein thevariable domain comprises a CDR having the sequence shown in SEQ IDNO:31 for CDRL1, the sequence shown in SEQ ID NO:32 for CDRL2 or thesequence shown in SEQ ID NO:33 for CDRL3.

The CDRs given in SEQ IDS NOS:28 and 30 to 34 referred to above arederived from a mouse monoclonal antibody hTNF40. However, SEQ ID NO:29consists of a hybrid CDR. The hybrid CDR comprises part of heavy chainCDR2 from mouse monoclonal antibody hTNF40 (SEQ ID NO:34) and part ofheavy chain CDR2 from a human group 3 germline V region sequence.

In one embodiment the antibody comprises a heavy chain wherein thevariable domain comprises a CDR having the sequence shown in SEQ IDNO:28 for CDRH1, the sequence shown in SEQ ID NO:29 or SEQ ID NO:34 forCDRH2 or the sequence shown in SEQ ID NO:30 for CDRH3 and a light chainwherein the variable domain comprises a CDR having the sequence shown inSEQ ID NO:31 for CDRL1, the sequence shown in SEQ ID NO:32 for CDRL2 orthe sequence shown in SEQ ID NO:33 for CDRL3.

In one embodiment the antibody comprises SEQ ID NO:28 for CDRH1, SEQ IDNO: 29 or SEQ ID NO:34 for CDRH2, SEQ ID NO:30 for CDRH3, SEQ ID NO:31for CDRL1, SEQ ID NO:32 for CDRL2 and SEQ ID NO:33 for CDRL3. Preferablythe antibody comprises SEQ ID NO:29 for CDRH2.

The anti-TNF antibody is preferably a CDR-grafted antibody molecule. Ina preferred embodiment the variable domain comprises human acceptorframework regions and non-human donor CDRs.

Preferably the antibody molecule has specificity for human TNF (formerlyknown as TNFα), wherein the light chain comprises the light chainvariable region of SEQ ID NO: 11 and the heavy chain comprises the heavychain variable region of SEQ ID NO: 12.

The anti-TNF antibody is preferably a Fab or Fab′ fragment.

Preferably the antibody molecule having specificity for human TNF is aFab′ and has a light chain sequence comprising or consisting of SEQ IDNO: 13 and a heavy chain sequence comprising or consisting of SEQ ID NO:14.

After expression, antibody fragments may be further processed, forexample by conjugation to another entity such as an effector molecule.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins (such as enzymatically activetoxins of bacterial or plant origin and fragments thereof e.g. ricin andfragments thereof) biologically active proteins, for example enzymes,other antibody or antibody fragments, synthetic or naturally occurringpolymers, nucleic acids and fragments thereof e.g. DNA, RNA andfragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy. Effector molecular may be attached to the antibody orfragment thereof by any suitable method, for example an antibodyfragment may be modified to attach at least one effector molecule asdescribed in WO05/003171 or WO05/003170 (the contents of which areincorporated herein by reference). WO05/003171 or WO05/003170 alsodescribe suitable effector molecules.

In one embodiment the antibody or fragment thereof, such as a Fab, isPEGylated to generate a product with the required properties, forexample similar to the whole antibodies, if required. For example, theantibody may be a PEGylated anti-TNF-α Fab′, as described inWO01/094585, preferably having attached to one of the cysteine residuesat the C-terminal end of the heavy chain a lysyl-maleimide-derived groupwherein each of the two amino groups of the lysyl residue has covalentlylinked to it a methoxypoly(ethyleneglycol) residue having a molecularweight of about 20,000 Da, such that the total average molecular weightof the methoxypoly(ethyleneglycol) residues is about 40,000 Da, morepreferably the lysyl-maleimide-derived group is[1-[[[2-[[3-(2,5-dioxo-1-pyrrolidinyl)-1-oxopropyl]amino]ethyl]amino]-carbonyl]-1,5-pentanediyl]bis(iminocarbonyl).

The cell may also comprise further polynucleotide sequences encoding oneor more further proteins of interest.

In one embodiment one or more E. coli host proteins that in the wildtype are known to co-purify with the recombinant protein of interestduring purification are selected for genetic modification, as describedin Humphreys et al. “Engineering of Escherichia coli to improve thepurification of periplasmic Fab′ fragments: changing the pI of thechromosomally encoded PhoS/PstS protein”, Protein Expression andPurification 37 (2004) 109-118 and WO04/035792 (the contents of whichare incorporated herein by reference). The use of such modified hostproteins improves the purification process for proteins of interest,especially antibodies, produced in E. coli by altering the physicalproperties of selected E. coli proteins so they no longer co-purify withthe recombinant antibody. Preferably the E. coli protein that is alteredis selected from one or more of Phosphate binding protein (PhoS/PstS),Dipeptide binding protein (DppA), Maltose binding protein (MBP) andThioredoxin.

In one embodiment a physical property of a contaminating host protein isaltered by the addition of an amino acid tag to the C-terminus orN-terminus. In a preferred embodiment the physical property that isaltered is the isoelectric point and the amino acid tag is apoly-aspartic acid tag attached to the C-terminus. In one embodiment theE. coli proteins altered by the addition of said tag are Dipeptidebinding protein (DppA), Maltose binding protein (MBP), Thioredoxin andPhosphate binding protein (PhoS/PstS). In one specific embodiment the pIof the E. coli Phosphate binding protein (PhoS/PstS) is reduced from 7.2to 5.1 by the addition of a poly-aspartic acid tag (polyD), containing 6aspartic acid residues to the C-terminus

Also preferred is the modification of specific residues of thecontaminating E. coli protein to alter its physical properties, eitheralone or in combination with the addition of N or C terminal tags. Suchchanges can include insertions or deletions to alter the size of theprotein or amino acid substitutions to alter pI or hydrophobicity. Inone embodiment these residues are located on the surface of the protein.In a preferred embodiment surface residues of the PhoS protein arealtered in order to reduce the pI of the protein. Preferably residuesthat have been implicated to be important in phosphate binding (Bass,U.S. Pat. No. 5,304,472) are avoided in order to maintain a functionalPhoS protein. Preferably lysine residues that project far out of thesurface of the protein or are in or near large groups of basic residuesare targeted. In one embodiment, the PhoS protein has a hexapoly-aspartic acid tag attached to the C-terminus whilst surfaceresidues at the opposite end of the molecule are targeted forsubstitution. Preferably selected lysine residues are substituted forglutamic acid or aspartic acid to confer a greater potential pI changethan when changing neutral residues to acidic ones. The designation fora substitution mutant herein consists of a letter followed by a numberfollowed by a letter. The first letter designates the amino acid in thewild-type protein. The number refers to the amino acid position wherethe amino acid substitution is being made, and the second letterdesignates the amino acid that is used to replace the wild-type aminoacid. In preferred mutations of PhoS in the present invention lysineresidues (K) 275, 107, 109, 110, 262, 265, 266, 309, 313 are substitutedfor glutamic acid (E) or glutamine (Q), as single or combined mutations,in addition lysine(K)318 may be substituted for aspartic acid (D) as asingle or combined mutation. Preferably the single mutations are K262E,K265E and K266E. Preferably the combined mutations are K265/266E andK110/265/266E. More preferably, all mutations are combined with thepolyaspartic acid (polyD) tag attached at the C-terminus and optionallyalso with the K318D substitution. In a preferred embodiment themutations result in a reduction in pI of at least 2 units. Preferablythe mutations of the present invention reduce the pI of PhoS from 7.2 tobetween about 4 and about 5.5. In one embodiment of the presentinvention the pI of the PhoS protein of E. coli is reduced from 7.2 toabout 4.9, about 4.8 and about 4.5 using the mutations polyD K318D,polyD K265/266E and polyD K110/265/266E respectively.

The recombinant gram-negative bacterial cell according to the presentinvention may be produced by any suitable means.

In the embodiments of the present invention wherein the chromosomal Tspgene is mutated and optionally one or more of the chromosomal spr gene,DegP gene, ptr gene and OmpT gene are mutated, the skilled person knowsof suitable techniques which may be used to replace a chromosomal genesequence with a mutated gene sequence. Suitable vectors may be employedwhich allow integration into the host chromosome by homologousrecombination. In the embodiment wherein the cell comprises two or threeof the above mutated genes, the mutated genes may be introduced into thegram-negative bacterium on the same or different vectors.

Suitable gene replacement methods are described, for example, inHamilton et al (New Method for Generating Deletions and GeneReplacements in Escherichia coli, Hamilton C. M. et al., Journal ofBacteriology September 1989, Vol. 171, No. 9 p 4617-4622), Skorupski etal (Positive selection vectors for allelic exchange, Skorupski K andTaylor R. K., Gene, 1996, 169, 47-52), Kiel et al (A general method forthe construction of Escherichia coli mutants by homologous recombinationand plasmid segregation, Kiel J. A. K. W. et al, Mol Gen Genet 1987,207:294-301), Blomfield et al (Allelic exchange in Escherichia coliusing the Bacillus subtilis sacB gene and a temperature sensitive pSC101replicon, Blomfield I. C. et al., Molecular Microbiology 1991, 5(6),1447-1457) and Ried et al. (An nptI-sacB-sacR cartridge for constructingdirected, unmarked mutations in Gram-negative bacteria by markerexchange-eviction mutagenesis, Ried J. L. and Collmer A., Gene 57 (1987)239-246). A suitable plasmid which enables homologousrecombination/replacement is the pKO3 plasmid (Link et al., 1997,Journal of Bacteriology, 179, 6228-6237).

The skilled person knows suitable techniques which may be used to insertthe recombinant polynucleotide encoding DsbC. The recombinantpolynucleotide encoding DsbC may be integrated into the cell's genomeusing a suitable vector such as the pKO3 plasmid.

Alternatively or additionally, the recombinant polynucleotide encodingDsbC may be non-integrated in a recombinant expression cassette. In oneembodiment an expression cassette is employed in the present inventionto carry the polynucleotide encoding DsbC which typically comprises aprotein coding sequence encoding DsbC and one or more regulatoryexpression sequences. The one or more regulatory expression sequencesmay include a promoter. The one or more regulatory expression sequencesmay also include a 3′ untranslated region such as a terminationsequence. Suitable promoters are discussed in more detail below.

In one embodiment an expression cassette is employed in the presentinvention to carry the polynucleotide encoding the protein of interestand/or the recombinant polynucleotide encoding DsbC which typicallycomprises one or more regulatory expression sequences, one or morecoding sequences encoding one or more proteins of interest and/or acoding sequence encoding DsbC. The one or more regulatory expressionsequences may include a promoter. The one or more regulatory expressionsequences may also include a 3′ untranslated region such as atermination sequence. Suitable promoters are discussed in more detailbelow.

In one embodiment, the cell according to the present invention comprisesone or more vectors, such as plasmid. The vector preferably comprisesone or more of the expression cassettes as defined above. In oneembodiment the polynucleotide sequence encoding a protein of interestand the polynucleotide encoding DsbC are inserted into one vector.Alternatively the polynucleotide sequence encoding a protein of interestand the polynucleotide encoding DsbC are inserted into separate vectors.

In the embodiment where the protein of interest is an antibodycomprising both heavy and light chains, the cell line may be transfectedwith two vectors, a first vector encoding a light chain polypeptide anda second vector encoding a heavy chain polypeptide. Alternatively, asingle vector may be used, the vector including sequences encoding lightchain and heavy chain polypeptides. Alternatively, the polynucleotidesequence encoding the antibody and the polynucleotide encoding DsbC areinserted into one vector. Preferably the vector comprises the sequencesencoding the light and heavy chain polypeptides of the antibody.

In the embodiment wherein the cell also expresses one or more furtherproteins as follows:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD; and/or    -   one or more protein capable of facilitating protein secretion or        translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and/or    -   one or more proteins capable of facilitating disulphide bond        formation, such as DsbA, DsbB, DsbD, DsbG;

the one or more further protein may be expressed from one or morepolynucleotides inserted into the same vector as the polynucleotideencoding DsbC and/or the polynucleotide sequence encoding a protein ofinterest. Alternatively the one or more polynucleotides may be insertedinto separate vectors.

The vector for use in the present invention may be produced by insertingone or more expression cassettes as defined above into a suitablevector. Alternatively, the regulatory expression sequences for directingexpression of the polynucleotide sequence may be contained in the vectorand thus only the encoding region of the polynucleotide may be requiredto complete the vector.

The polynucleotide encoding DsbC and/or the polynucleotide encoding theprotein of interest is suitably inserted into a replicable vector,typically an autonomously replicating vector, for expression in the cellunder the control of a suitable promoter for the cell. Many vectors areknown in the art for this purpose and the selection of the appropriatevector may depend on the size of the nucleic acid and the particularcell type.

Examples of vectors which may be employed to transform the host cellwith a polynucleotide according to the invention include:

-   -   a plasmid, such as pBR322 or pACYC184, and/or    -   a viral vector such as bacterial phage    -   a transposable genetic element such as a transposon

Such vectors usually comprise a plasmid origin of DNA replication, anantibiotic selectable marker, a promoter and transcriptional terminatorseparated by a multi-cloning site (expression cassette) and a DNAsequence encoding a ribosome binding site.

The promoters employed in the present invention can be linked to therelevant polynucleotide directly or alternatively be located in anappropriate position, for example in a vector such that when therelevant polypeptide is inserted the relevant promoter can act on thesame. In one embodiment the promoter is located before the encodingportion of the polynucleotide on which it acts, for example a relevantpromoter before each encoding portion of polynucleotide. “Before” asused herein is intended to imply that the promoter is located at the 5prime end in relation to the encoding polynucleotide portion.

The promoters may be endogenous or exogenous to the host cells. Suitablepromoters include Lac, tac, trp, PhoA, Ipp, Arab, Tet and T7.

One or more promoters employed may be inducible promoters.

In the embodiment wherein the polynucleotide encoding DsbC and thepolynucleotide encoding the protein of interest are inserted into onevector, the nucleotide sequences encoding DsbC and the protein ofinterest may be under the control of a single promoter or separatepromoters. In the embodiment wherein the nucleotide sequences encodingDsbC and the protein of interest are under the control of separatepromoters, the promoter may be independently inducible promoters.

Promoters for use in bacterial systems also generally contain aShine-Dalgamo (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA.

In the embodiments of the present invention wherein a polynucleotidesequence comprises two or more encoding sequences for two or moreproteins of interest, for example an antibody light chain and antibodyheavy chain, the polynucleotide sequence may comprise one or moreinternal ribosome entry site (IRES) sequences which allows translationinitiation in the middle of an mRNA. An IRES sequence may be positionedbetween encoding polynucleotide sequences to enhance separatetranslation of the mRNA to produce the encoded polypeptide sequences.

The expression vector preferably also comprises a dicistronic messagefor producing the antibody or antigen binding fragment thereof asdescribed in WO 03/048208 or WO2007/039714 (the contents of which areincorporated herein by reference). Preferably the upstream cistroncontains DNA coding for the light chain of the antibody and thedownstream cistron contains DNA coding for the corresponding heavychain, and the dicistronic intergenic sequence (IGS) preferablycomprises a sequence selected from IGS1 (SEQ ID NO: 36), IGS2 (SEQ IDNO: 37), IGS3 (SEQ ID NO: 38) and IGS4 (SEQ ID NO: 39).

The terminators may be endogenous or exogenous to the host cells. Asuitable terminator is rrnB.

Further suitable transcriptional regulators including promoters andterminators and protein targeting methods may be found in “Strategiesfor Achieving High-Level Expression of Genes in Escherichia coli” SavvasC. Makrides, Microbiological Reviews, September 1996, p 512-538.

The DsbC polynucleotide inserted into the expression vector preferablycomprises the nucleic acid encoding the DsbC signal sequence and theDsbC coding sequence. The vector preferably contains a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells, preferably to replicate independently of the hostchromosome. Such sequences are well known for a variety of bacteria.

In one embodiment the DsbC and/or the protein of interest comprises ahistidine-tag at the N-terminus and/or C-terminus

The antibody molecule may be secreted from the cell or targeted to theperiplasm by suitable signal sequences. Alternatively, the antibodymolecules may accumulate within the cell's cytoplasm. Preferably theantibody molecule is targeted to the periplasm.

The polynucleotide encoding the protein of interest may be expressed asa fusion with another polypeptide, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature polypeptide. The heterologous signal sequence selected should beone that is recognized and processed by the host cell. For prokaryotichost cells that do not recognize and process the native or a eukaryoticpolypeptide signal sequence, the signal sequence is substituted by aprokaryotic signal sequence. Suitable signal sequences include OmpA,PhoA, LamB, PelB, DsbA and DsbC.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

In a preferred embodiment of the present invention the present inventionprovides a multi-cistronic vector comprising the polynucleotide sequenceencoding DsbC and the polynucleotide sequence encoding a protein ofinterest. The multicistronic vector may be produced by an advantageouscloning method which allows repeated sequential cloning ofpolynucleotide sequences into a vector. The method uses compatiblecohesive ends of a pair of restrictions sites, such as the “AT” ends ofAse I and Nde I restriction sites. A polynucleotide sequence comprisinga coding sequence and having compatible cohesive ends, such as aAseI-NdeI fragment, may be cloned into a restrictions site in thevector, such as Nde I. The insertion of the polynucleotide sequencedestroys the 5′ restriction site but creates a new 3′ restriction site,such as NdeI, which may then be used to insert a further polynucleotidesequence comprising compatible cohesive ends. The process may then berepeated to insert further sequences. Each polynucleotide sequenceinserted into the vector comprises non-coding sequence 3′ to the stopcodon which may comprise an Ssp I site for screening, a Shine Dalgarnoribosome binding sequence, an A rich spacer and an NdeI site encoding astart codon.

A diagrammatic representation of the creation of a vector comprising apolynucleotide sequence encoding a light chain of an antibody (LC), aheavy chain of an antibody (HC), a DsbC polynucleotide sequence and afurther polynucleotide sequence is shown in FIG. 6.

Successfully mutated strains may be identified using methods well knownin the art including colony PCR DNA sequencing and colony PCRrestriction enzyme mapping.

Embodiments of the invention described herein with reference to thepolynucleotide apply equally to alternative embodiments of theinvention, for example vectors, expression cassettes and/or host cellscomprising the components employed therein, as far as the relevantaspect can be applied to same.

According to a third aspect of the present invention there is provided amethod for producing a recombinant protein of interest comprisingexpressing the recombinant protein of interest in a recombinantgram-negative bacterial cell as described above in the first or secondaspect of the present invention. The method for producing a recombinantprotein of interest comprising:

a. culturing a recombinant gram-negative bacterial cell as defined abovein a culture medium under conditions effective to express therecombinant protein of interest and the recombinant polynucleotideencoding DsbC; and

b. recovering the recombinant protein of interest from the periplasm ofthe recombinant gram-negative bacterial cell and/or the culture medium.

The gram negative bacterial cell and protein of interest preferablyemployed in the method of the present invention are described in detailabove.

When the polynucleotide encoding the protein of interest is exogenousthe polynucleotide may be incorporated into the host cell using anysuitable means known in the art. As discussed above, typically thepolynucleotide is incorporated as part of an expression vector which istransformed into the cell. Accordingly, in one aspect the cell accordingto the present invention comprises an expression cassette comprising thepolynucleotide encoding the protein of interest and an expressioncassette comprising the polynucleotide encoding DsbC.

The polynucleotide sequence encoding the protein of interest and thepolynucleotide sequence encoding DsbC can be transformed into a cellusing standard techniques, for example employing rubidium chloride, PEGor electroporation.

The method according to the present invention may also employ aselection system to facilitate selection of stable cells which have beensuccessfully transformed with the polynucleotide encoding the protein ofinterest. The selection system typically employs co-transformation of apolynucleotide sequence encoding a selection marker. In one embodiment,each polynucleotide transformed into the cell further comprises apolynucleotide sequence encoding one or more selection markers.Accordingly, the transformation of the polynucleotides encoding DsbC andthe protein of interest and the one or more polynucleotides encoding themarker occurs together and the selection system can be employed toselect those cells which produce the desired proteins.

Cells able to express the one or more markers are able tosurvive/grow/multiply under certain artificially imposed conditions, forexample the addition of a toxin or antibiotic, because of the propertiesendowed by the polypeptide/gene or polypeptide component of theselection system incorporated therein (e.g. antibiotic resistance).Those cells that cannot express the one or more markers are not able tosurvive/grow/multiply in the artificially imposed conditions. Theartificially imposed conditions can be chosen to be more or lessvigorous, as required.

Any suitable selection system may be employed in the present invention.Typically the selection system may be based on including in the vectorone or more genes that provides resistance to a known antibiotic, forexample a tetracycline, chloramphenicol, kanamycin or ampicillinresistance gene. Cells that grow in the presence of a relevantantibiotic can be selected as they express both the gene that givesresistance to the antibiotic and the desired protein.

In one embodiment, the method according to the present invention furthercomprises the step of culturing the transformed cell in a medium tothereby express the protein of interest.

An inducible expression system or a constitutive promoter may be used inthe present invention to express the protein of interest and/or theDsbC. Suitable inducible expression systems and constitutive promotersare well known in the art.

In one embodiment wherein the polynucleotide encoding DsbC and thepolynucleotide encoding the protein of interest are under the control ofthe same or separate inducible promoters, the expression of thepolynucleotide sequence encoding a protein of interest and therecombinant polynucleotide encoding DsbC is induced by adding an inducerto the culture medium.

Any suitable medium may be used to culture the transformed cell. Themedium may be adapted for a specific selection system, for example themedium may comprise an antibiotic, to allow only those cells which havebeen successfully transformed to grow in the medium.

The cells obtained from the medium may be subjected to further screeningand/or purification as required. The method may further comprise one ormore steps to extract and purify the protein of interest as required.

The polypeptide may be recovered from the strain, including from thecytoplasm, periplasm, or supernatant.

The specific method (s) used to purify a protein depends on the type ofprotein. Suitable methods include fractionation on immuno-affinity orion-exchange columns; ethanol precipitation; reversed-phase HPLC;hydrophobic-interaction chromatography; chromatography on silica;chromatography on an ion-exchange resin such as S-SEPHAROSE and DEAE;chromatofocusing; ammonium-sulfate precipitation; and gel filtration.

In one embodiment the method further comprises separating therecombinant protein of interest from DsbC.

Antibodies may be suitably separated from the culture medium and/orcytoplasm extract and/or periplasm extract by conventional antibodypurification procedures such as, for example, protein A-Sepharose,protein G chromatography, protein L chromatography, thiophilic, mixedmode resins, His-tag, FLAGTag, hydroxylapatite chromatography, gelelectrophoresis, dialysis, affinity chromatography, Ammonium sulphate,ethanol or PEG fractionation/precipitation, ion exchange membranes,expanded bed adsorption chromatography (EBA) or simulated moving bedchromatography.

The method may also include a further step of measuring the quantity ofexpression of the protein of interest and selecting cells having highexpression levels of the protein of interest.

The method may also including one or more further downstream processingsteps such as PEGylation of the protein of interest, such as an antibodyor antibody fragment.

One or more method steps described herein may be performed incombination in a suitable container such as a bioreactor.

EXAMPLES

Cell Lines

For all experiments the E. coli cell line W3110 was used as the parentalwild-type cell line.

Cell lines were created carrying the following mutations:

-   -   a. a mutated Tsp gene;    -   b. a mutated Tsp gene and carrying recombinant DsbC;    -   c. a mutated Tsp gene and a mutated spr gene;    -   d. a mutated Tsp gene and a mutated spr gene and carrying        recombinant DsbC.

Example 1 Generation of Cell Line Carrying Mutated Tsp Gene MXE001(ΔTsp)

The MXE001 strain was generated as follows:

The Tsp cassette was moved as Sal I, Not I restriction fragments intosimilarly restricted pKO3 plasmids. The pKO3 plasmid uses thetemperature sensitive mutant of the pSC101 origin of replication (RepA)along with a chloramphenicol marker to force and select for chromosomalintegration events. The sacB gene which encodes for levansucrase islethal to E. coli grown on sucrose and hence (along with thechloramphenicol marker and pSC101 origin) is used to force and selectfor de-integration and plasmid curing events. This methodology had beendescribed previously (Hamilton et al., 1989, Journal of Bacteriology,171, 4617-4622 and Blomfield et al., 1991, Molecular Microbiology, 5,1447-1457). The pKO3 system removes all selective markers from the hostgenome except for the inserted gene.

The following plasmids were constructed.

pMXE191 comprising the knockout mutated Tsp gene as shown in the SEQ IDNO: 3 comprising EcoR I and Ase I restriction markers.

The plasmid was then transformed into electro-competent competent E.coli W3110 cells prepared using the method found in Miller, E. M. andNickoloff, J. A., “Escherichia coli electrotransformation,” in Methodsin Molecular Biology, vol. 47, Nickoloff, J. A. (ed.), Humana Press,Totowa, N.J., 105 (1995).

Day 1 40 μl of E. coli cells were mixed with (10 pg) 1 μl of pKO3 DNA ina chilled BioRad 0.2 cm electroporation cuvette before electroporationat 2500V, 25 μF and 200 Ω. 1000 μl of 2×PY was added immediately, thecells recovered by shaking at 250 rpm in an incubator at 30° C. for 1hour. Cells were serially 1/10 diluted in 2×PY before 100 μl aliquotswere plated out onto 2×PY agar plates containing chloramphenicol at 20μg/ml prewarmed at 30° C. and 43° C. Plates were incubated overnight at30° C. and 43° C.

Day 2 The number of colonies grown at 30° C. gave an estimate of theefficiency of electroporation whilst colonies that survive growth at 43°C. represent potential integration events. Single colonies from the 43°C. plate were picked and resuspended in 10 ml of 2×PY. 100 μl of thiswas plated out onto 2×PY agar plates containing 5% (w/v) sucrosepre-warmed to 30° C. to generate single colonies. Plates were incubatedovernight at 30° C.

Day 3 Colonies here represent potential simultaneous de-integration andplasmid curing events. If the de-integration and curing events happenedearly on in the growth, then the bulk of the colony mass will be clonal.Single colonies were picked and replica plated onto 2×PY agar thatcontained either chloramphenicol at 20 μg/ml or 5% (w/v) sucrose. Plateswere incubated overnight at 30° C.

Day 4 Colonies that both grow on sucrose and die on chloramphenicolrepresent potential chromosomal replacement and plasmid curing events.These were picked and screened by PCR with a mutation specificoligonucleotide. Colonies that generated a positive PCR band of thecorrect size were struck out to produce single colonies on 2×PY agarcontaining 5% (w/v) sucrose and the plates were incubated overnight at30° C.

Day 5 Single colonies of PCR positive, chloramphenicol sensitive andsucrose resistant E. coli were used to make glycerol stocks, chemicallycompetent cells and act as PCR templates for a PCR reaction with 5′ and3′ flanking oligos to generate PCR product for direct DNA sequencingusing Taq polymerase.

Cell strain MXE001 was tested to confirm successful modification ofgenomic DNA carrying the mutated Tsp gene by PCR amplification of theregion of the Tsp gene comprising a non-naturally occurring Ase Irestriction site, as shown in FIGS. 1 a, 1 b and 1 c, usingoligonucleotides primers. The amplified regions of the DNA were thenanalyzed by gel electrophoresis before and after incubation with Ase Irestriction enzyme to confirm the presence of the non-naturallyoccurring Ase I restriction site in the mutated genes. This method wascarried out as follows:

The following oligos were used to amplify, using PCR, genomic DNA fromprepared E. coli cell lysates from MXE001 and W3110:

(SEQ ID NO: 15) 6284 Tsp 3′ 5′-GCATCATAATTTTCTTTTTACCTC-3′(SEQ ID NO: 16) 6283 Tsp 5′ 5′-GGGAAATGAACCTGAGCAAAACGC-3′

The lysates were prepared by heating a single colony of cells for 10minutes at 95° C. in 20 μl of 1×PCR buffer. The mixture was allowed tocool to room temperature then centrifugation at 13,200 rpm for 10minutes. The supernatant was removed and labeled as ‘cell lysate’.

Each strain was amplified using the Tsp oligos pair.

The DNA was amplified using a standard PCR procedure.

  5 ul Buffer ×10 (Roche)   1 ul dNTP mix (Roche, 10 mM mix) 1.5 ul 5′oligo (5 pmol) 1.5 ul 3′ oligo (5 pmol)   2 ul Cell lysate 0.5 ul TaqDNA polymerase (Roche 5 U/ul) 38.5 ul  H2O

PCR cycle.

94° C. 1 minute 94° C. 1 minute 55° C. 1 minute {close oversize brace}repeated for 30 cycles 72° C. 1 minute 72° C. 10 minutes

Once the reactions were complete 25 ul was removed to a new microfugetube for digestion with Ase I. To the 25 ul of PCR reaction 19 ul ofH2O, 5 ul of buffer 3 (NEB), 1 ul of Ase I (NEB) was added, mixed andincubated at 37° C. for 2 hours.

To the remaining PCR reaction 5 ul of loading buffer (×6) was added and20 ul was loaded onto a 0.8% TAE 200 ml agarose gel (Invitrogen) plusEthidium Bromide (5 ul of 10 mg/ml stock) and run at 100 volts for 1hour. 10 ul of size marker (Perfect DNA marker 0.1-12 Kb, Novagen) wasloaded in the final lane.

Once the Ase I digestions were complete 10 ul of loading buffer (×6) wasadded and 20 ul was loaded onto a 0.8% TAE agarose gel (Invitrogen) plusEthidium Bromide (5 ul of 10 mg/ml stock) and run at 100 volts for 1hour. 10 ul of size marker (Perfect DNA marker 0.1-12 Kb, Novagen) wasloaded in the final lane. Both gels were visualized using UVtransluminator.

The genomic fragment amplified showed the correct sized band of 2.8 Kbfor Tsp. Following digestion with Ase I this confirmed the presence ofthe introduced Ase I sites in the Tsp deficient strain MXE001 but not inthe W3110 control.

MXE001: genomic DNA amplified using the Tsp primer set and the resultingDNA was digested with Ase Ito produce 2.2 and 0.6 Kbps bands.

W3110 PCR amplified DNA was not digested by Ase I restriction enzyme.

Example 2 Generation of Cell Lines Carrying Mutated Spr Gene and CellLines Carrying Mutated Tsp Gene and Mutated Spr Gene

The spr mutations were generated and selected for using acomplementation assay.

The spr gene was mutated using the Clontech® random mutagenesisdiversity PCR kit which introduced 1 to 2 mutations per 1000 bp. Themutated spr PCR DNA was cloned into an inducible expression vector [pTTOCDP870] which expresses CDP870 Fab′ along with the spr mutant. Thisligation was then electro-transformed into an E. coli strain MXE001(ΔTsp) prepared using the method found in Miller, E. M. and Nickoloff,J. A., “Escherichia coli electrotransformation,” in Methods in MolecularBiology, vol. 47, Nickoloff, J. A. (ed.), Humana Press, Totowa, N.J.,105 (1995). The following protocol was used, 40 ul of electro competentMXE001, 2.5 ul of the ligation (100 pg of DNA) was added to a 0.2 cmelectroporation cuvette, electro-transformation was performed using asBioRad Genepulser Xcell with the following conditions, 2500V, 25 μF and200 Ω. After the electro-transformation 1 ml of SOC (Invitrogen)(pre-warmed to 37° C.) was added and the cells left to recover at 37° C.for 1 hour with gentle agitation.

The cells where plated onto Hypotonic agar (5 g/L Yeast extract, 2.5 g/LTryptone, 15 g/L Agar (all Difco)) and incubated at 40° C. Cells whichformed colonies were re-plated onto HLB at 43° C. to confirm restorationof the ability to grow under low osmotic conditions at high temperatureto the MXE001 strain. Plasmid DNA was prepared from the selected clonesand sequenced to identify spr mutations.

Using this method eight single, one double mutation and two multiplemutations in the spr protein were isolated which complemented the ΔTspphenotype as follows:

-   -   1. V98E    -   2. D133A    -   3. V135D    -   4. V135G    -   5. G147C    -   6. S95F and Y115F    -   7. I70T    -   8. N31T, Q73R, R100G, G140C    -   9. R62C, Q99P, R144C    -   10. L108S    -   11. L136P

The individual mutations 1 to 5 identified above and three catalytictriad mutations of spr (C94A, H145A, H157A) and W174R were used togenerate new strains using either the wild-type W3110 E. coli strain(genotype: F- LAM- IN (rrnD-rrnE)1 rph1 (ATCC no. 27325)) to create sprmutated strains or MXE001 strain from Example 1 to make combinedΔTsp/mutant spr strains.

The following mutant E. coli cell strains were generated using a genereplacement vector system using the pKO3 homologousrecombination/replacement plasmid (Link et al., 1997, Journal ofBacteriology, 179, 6228-6237), as described in Example 1 for thegeneration of MXE001.

TABLE 1 Mutant E. coli Cell Strain Genotype Spr Vectors MXE001 ΔTsp —MXE008 ΔTsp, spr D133A pMXE339, pK03 spr D133A (-SalI) MXE009 ΔTsp, sprH157A pMXE345, pK03 spr H157A (-SalI) MXE010 spr G147C pMXE338, pK03 sprG147C (-SalI) MXE011 spr C94A pMXE343, pK03 spr C94A (-SalI) MXE012 sprH145A pMXE344, pK03 spr H145A (-SalI) MXE013 spr W174R pMXE346, pK03 sprW174R (-SalI) MXE014 ΔTsp, spr V135D pMXE340, pK03 spr V135D (-SalI)MXE015 ΔTsp, spr V98E pMXE342, pK03 spr V98E (-SalI) MXE016 ΔTsp, sprC94A pMXE343, pK03 spr C94A (-SalI) MXE017 ΔTsp, spr H145A pMXE344, pK03spr H145A (-SalI) MXE018 ΔTsp, spr V135G pMXE341, pK03 spr V135G (-SalI)

The mutant spr integration cassettes were moved as Sal I, Not Irestriction fragments into similarly restricted pKO3 plasmids.

The plasmid uses the temperature sensitive mutant of the pSC101 originof replication (RepA) along with a chloramphenicol marker to force andselect for chromosomal integration events. The sacB gene which encodesfor levansucrase is lethal to E. coli grown on sucrose and hence (alongwith the chloramphenicol marker and pSC101 origin) is used to force andselect for de-integration and plasmid curing events. This methodologyhad been described previously (Hamilton et al., 1989, Journal ofBacteriology, 171, 4617-4622 and Blomfield et al., 1991, MolecularMicrobiology, 5, 1447-1457). The pKO3 system removes all selectivemarkers from the host genome except for the inserted gene.

The pK03 vectors listed below were constructed, comprising the mutatedspr genes including a silent mutation within the spr sequence whichremoves a SalI restriction site for clone identification.

-   pMXE336, pK03 spr S95F (−SalI)-   pMXE337, pK03 spr Y115F (−SalI)-   pMXE338, pK03 spr G147C (−SalI)-   pMXE339, pK03 spr D133A (−SalI)-   pMXE340, pK03 spr V135D (−SalI)-   pMXE341, pK03 spr V135G (−SalI)-   pMXE342, pK03 spr V98E (−SalI)-   pMXE343, pK03 spr C94A (−SalI)-   pMXE344, pK03 spr H145A (−SalI)-   pMXE345, pK03 spr H157A (−SalI)-   pMXE346, pK03 spr W174R (−SalI)

These plasmids were then transformed into chemically competent E. coliW3110 cells prepared using the method found in Miller, E. M. andNickoloff, J. A., “Escherichia coli electrotransformation,” in Methodsin Molecular Biology, vol. 47, Nickoloff, J. A. (ed.), Humana Press,Totowa, N.J., 105 (1995) or into MXE001 strain from Example 1 to makecombined ΔTsp/mutant spr strains, as shown in Table 1.

Day 1 40 μl of electro-competent E. coli cells or MXE001 cells weremixed with (10 pg) 1 μl of pKO3 DNA in a chilled BioRad 0.2 cmelectroporation cuvette before electroporation at 2500V, 25 μF and 200Ω. 1000 μl of 2×PY was added immediately, the cells recovered by shakingat 250 rpm in an incubator at 30° C. for 1 hour. Cells were serially1/10 diluted in 2×PY before 100 μl aliquots were plated out onto 2×PYagar plates containing chloramphenicol at 20 μg/ml prewarmed at 30° C.and 43° C. Plates were incubated overnight at 30° C. and 43° C.

Day 2 The number of colonies grown at 30° C. gave an estimate of theefficiency of electroporation whilst colonies that survive growth at 43°C. represent potential integration events. Single colonies from the 43°C. plate were picked and resuspended in 10 ml of 2×PY. 100 μl of thiswas plated out onto 2×PY agar plates containing 5% (w/v) sucrosepre-warmed to 30° C. to generate single colonies. Plates were incubatedovernight at 30° C.

Day 3 Colonies here represent potential simultaneous de-integration andplasmid curing events. If the de-integration and curing events happenedearly on in the growth, then the bulk of the colony mass will be clonal.Single colonies were picked and replica plated onto 2×PY agar thatcontained either chloramphenicol at 20 μg/ml or 5% (w/v) sucrose. Plateswere incubated overnight at 30° C.

Day 4 Colonies that both grow on sucrose and die on chloramphenicolrepresent potential chromosomal replacement and plasmid curing events.These were picked and screened by PCR plus restriction digest for theloss of a SalI site. Colonies that generated a positive PCR band of thecorrect size and resistance to digestion by SalI were struck out toproduce single colonies on 2×PY agar containing 5% (w/v) sucrose and theplates were incubated overnight at 30° C.

Day 5 Single colonies of PCR positive, chloramphenicol sensitive andsucrose resistant E. coli were used to make glycerol stocks, chemicallycompetent cells and act as PCR templates for a PCR reaction with 5′ and3′ flanking oligos to generate PCR product for direct DNA sequencingusing Taq polymerase to confirm the correct mutation.

Example 3 Generation of Plasmid for Fab′ and DsbC Expression

A plasmid was constructed containing both the heavy and light chainsequences of an anti-TNF Fab′ and the sequence encoding DsbC.

A dicistronic message was created of the anti-TNFα Fab′ fragment(referred to as CDP870) described in WO01/94585. The upstream cistronencoded the light chain of the antibody (SEQ ID NO: 13) whilst thedownstream cistron encoded the heavy chain of the antibody (SEQ ID NO:14). A DNA sequence encoding the OmpA signal peptide was fused to the 5′end of the DNA coding for each of the light chain and the heavy chain toallow efficient secretion to the periplasm. The intergenic sequence(IGS) 2 was used as shown in SEQ ID NO: 37.

Plasmid pDPH358 (pTTO 40.4 CDP870 IGS2), an expression vector for theCDP870 Fab′ (an anti-TNF Fab′) and DsbC (a periplasmic polypeptide), wasconstructed using conventional restriction cloning methodologies whichcan be found in Sambrook et al 1989, Molecular cloning: a laboratorymanual. CSHL press, N.Y. The plasmid pDPH358 contained the followingfeatures; a strong tac promoter and lac operator sequence. As shown inFIG. 6, the plasmid contained a unique EcoRI restriction site after thecoding region of the Fab′ heavy chain, followed by a non-coding sequenceand then a unique NdeI restriction site. The DsbC gene was PCR clonedusing W3110 crude chromosomal DNA as a template such that the PCRproduct encoded for a 5′ EcoRI site followed by a strong ribosomebinding, followed by the native start codon, signal sequence and maturesequence of DsbC, terminating in a C-terminal His tag and finally anon-coding NdeI site. The EcoRI-NdeI PCR fragment was restricted andligated into the expression vector such that all three polypeptides:Fab′ light chain, Fab′ heavy chain and DsbC were encoded on a singlepolycistronic mRNA.

The Fab light chain, heavy chain genes and DcbC gene were transcribed asa single polycistronic message. DNA encoding the signal peptide from theE. coli OmpA protein was fused to the 5′ end of both light and heavychain gene sequences, which directed the translocation of thepolypeptides to the E. coli periplasm. Transcription was terminatedusing a dual transcription terminator rrnB t1t2. The laclq gene encodedthe constitutively expressed Lac I repressor protein. This repressedtranscription from the tac promoter until de-repression was induced bythe presence of allolactose or IPTG. The origin of replication used wasp15A, which maintained a low copy number. The plasmid contained atetracycline resistance gene for antibiotic selection.

Example 4 Expression of Anti-TNF Fab′ and DsbC in the Mutant Cell Lines

Expression of Anti-TNF Fab′ and DsbC

The MXE001 strain provided in Example 1 and the mutant strains, MXE008and MXE009 provided in Example 2 were transformed with the plasmidgenerated in Example 3.

The transformation of the strains was carried out using the method foundin Chung C. T et al Transformation and storage of bacterial cells in thesame solution. PNAS 86:2172-2175 (1989).

Expression of Anti-TNF Fab′

The strains MXE001, MXE008 and MXE009 were transformed with plasmidpMXE117 (pTTO CDP870 or 40.4 IGS2), an expression vector for the CDP870Fab′ (an anti-TNF Fab′ having a light chain sequence shown in SEQ ID NO:13 and a heavy chain sequence shown in SEQ ID NO: 14), as described inExample 3, which was constructed using conventional restriction cloningmethodologies which can be found in Sambrook et al 1989, Molecularcloning: a laboratory manual. CSHL press, N.Y. The plasmid pMXE117 (pTTOCDP870 or 40.4 IGS2) contained the following features; a strong tacpromoter and lac operator sequence. The Fab light and heavy chain geneswere transcribed as a single dicistronic message. DNA encoding thesignal peptide from the E. coli OmpA protein was fused to the 5′ end ofboth light and heavy chain gene sequences, which directed thetranslocation of the polypeptides to the E. coli periplasm.Transcription was terminated using a dual transcription terminator rrnBt1t2. The laclq gene encoded the constitutively expressed Lac Irepressor protein. This repressed transcription from the tac promoteruntil de-repression was induced by the presence of allolactose or IPTG.The origin of replication used was p15A, which maintained a low copynumber. The plasmid contained a tetracycline resistance gene forantibiotic selection.

The transformation of the strains was carried out using the method foundin Chung C. T et al Transformation and storage of bacterial cells in thesame solution. PNAS 86:2172-2175 (1989).

Example 5 Growth of Mutated E. Coli Strains and Expression of Anti-TNFFab′ in Mutated E. Coli Strains Using High Density Fermentations

The following strains produced in Example 4 were tested in fermentationexperiments comparing growth and expression of an anti-TNFα Fab′:

-   MXE001 expressing anti-TNF Fab′-   MXE008 expressing anti-TNF Fab′-   MXE009 expressing anti-TNF Fab′-   MXE001 expressing anti-TNF Fab′ and DsbC-   MXE008 expressing anti-TNF Fab′ and DsbC-   MXE009 expressing anti-TNF Fab′ and DsbC    Growth Medium.

The fermentation growth medium was based on SM6E medium (described inHumphreys et al., 2002, Protein Expression and Purification, 26,309-320) with 3.86 g/l NaH₂PO₄.H₂O and 112 g/l glycerol.

Inoculum. Inoculum cultures were grown in the same medium supplementedwith 10 μg/ml tetracycline. Cultures were incubated at 30° C. withagitation for approximately 22 hours.

Fermentation. Fermenters (2.5 litres total volume) were seeded withinoculum culture to 0.3-0.5 OD₆₀₀. Temperature was maintained at 30° C.during the growth phase and was reduced to 25° C. prior to induction.The dissolved oxygen concentration was maintained above 30% airsaturation by variable agitation and airflow. Culture pH was controlledat 7.0 by automatic titration with 15% (v/v) NH₄OH and 10% (v/v) conc.H₂SO₄. Foaming was controlled by the addition of 10% (v/v) Struktol J673solution (Schill and Seilacher). A number of additions were made atdifferent stages of the fermentation. When biomass concentration reachedapproximately 40 OD₆₀₀, magnesium salts and NaH₂PO₄.H₂O were added.Further additions of NaH₂PO₄.H₂O were made prior to and during theinduction phase to ensure phosphate was maintained in excess. When theglycerol present at the beginning of fermentation had depleted(approximately 75 OD₆₀₀) a continuous feed of 80% (w/w) glycerol wasapplied. At the same point in the fermentation an IPTG feed at 170 μMwas applied. The start of IPTG feeding was taken as the start ofinduction. Fermentations were typically run for 64-120 hours at glycerolfeed rates (ranging between 0.5 and 2.5 ml/h).

Measurement of biomass concentration and growth rate. Biomassconcentration was determined by measuring the optical density ofcultures at 600 nm.

Periplasmic Extraction. Cells were collected from culture samples bycentrifugation. The supernatant fraction was retained (at −20° C.) forfurther analysis. The cell pellet fraction was resuspended to theoriginal culture volume in extraction buffer (100 mM Tris-HCl, 10 mMEDTA; pH 7.4). Following incubation at 60° C. for approximately 16 hoursthe extract was clarified by centrifugation and the supernatant fractionretained (at −20° C.) for analysis.

Fab′ quantification. Fab′ concentrations in periplasmic extracts andculture supernatants were determined by Fab′ assembly ELISA as describedin Humphreys et al., 2002, Protein Expression and Purification, 26,309-320 and using Protein G hplc. A HiTrap Protein-G HP 1 ml column(GE-Healthcare or equivalent) was loaded with analyte (approximatelyneutral pH, 30° C., 0.2 um filtered) at 2 ml/min, the column was washedwith 20 mM phosphate, 50 mM NaCl pH 7.4 and then Fab′ eluted using aninjection of 50 mM Glycine/HCl pH 2.7. Eluted Fab′ was measured by A280on a Agilent 1100 or 1200 HPLC system and quantified by reference to astandard curve of a purified Fab′ protein of known concentration.

FIG. 1 shows the growth profile of anti-TNF Fab′ expressing strainsMXE001 and MXE008 and the growth profile of anti-TNFα Fab′ andrecombinant DsbC expressing strains MXE001 and MXE008. It can be seenthat the strains expressing DsbC exhibit improved growth compared to thecorresponding cell strains which do not express recombinant DsbC. It canalso be seen that the presence of the spr mutation in the strainsimproves cell growth.

FIG. 2 shows Fab yield from the periplasm (shaded symbols) andsupernatant (open unshaded symbols) from anti-TNF Fab′ and recombinantDsbC expressing E. coli strains MXE001, MXE008 and MXE009. It can beseen from this graph that all three strains produced a high yield ofanti-TNFα Fab′ with strains MXE008 and MXE009 producing over 2.2 g/Lperiplasmic anti-TNFα Fab′ in 40 hours. It can also be seen that MXE008and MXE009 strains carrying a mutant spr gene exhibited reduced lysiscompared to MXE001 which can be seen as less supernatant anti-TNFα Fab′(open symbols).

FIG. 3 shows Fab yield from the periplasm (shaded symbols) andsupernatant (open unshaded symbols) from anti-TNFα Fab′ expressing E.coli strains MXE001 and MXE008 and from anti-TNFα Fab′ and recombinantDsbC expressing E. coli strains MXE001 and MXE008. It can be seen fromthis graph that the strains expressing recombinant DsbC produced a highyield of anti-TNFα Fab′ with strain MXE008 producing 2.4 g/L periplasmicanti-TNFα Fab′ in 40 hours. It can also be seen that the MXE008 strainscarrying a mutant spr gene exhibited reduced lysis compared to theMXE001 strains which can be seen as less supernatant anti-TNFα Fab′(open symbols).

Example 6 Growth of Mutated E. Coli Strains and Expression of Fab A andFab B in Mutated E. Coli Strains Using High Density Fermentations

The effect on the yield of protein production from a cell of the presentinvention was also carried out for two further Fab proteins, Fab Ahaving specificity for antigen A and Fab B having specificity forantigen B, using the same method described above in Example 5 andcompared to W3110 strains expressing Fab A and Fab B.

FIG. 4 shows total Fab yield from the periplasm from Fab A and Fab Bexpressing E. coli strain W3110 and from Fab A and recombinant DsbC orFab B and recombinant DsbC expressing E. coli strain MXE008. It can beseen from this graph that the strains expressing recombinant DsbCproduced a significantly higher yield of Fab A and Fab B in MXE008compared to W3110.

Example 7 Growth of E. Coli Strains Expressing Anti-TNF Fab′ and DsbCUsing Large Scale Fermentations

The following strain, as produced by example 4 was tested infermentation experiments comparing growth of the strain and theexpression of an anti-TNFα Fab′:

MXE008 expressing anti-TNF Fab′ and DsbC

The fermentations were carried out as follows:

The MXE008 expressing anti-TNF Fab′ and DsbC cells were grown initiallyusing a complex medium of yeast extract and peptone in shake flaskculture. The cells were then transferred to a seed stage fermenter usinga chemically defined medium. The cells were grown under non-nutrientlimiting conditions until a defined transfer point. The cells were thentransferred to a 5 L or 250 L production fermenter using a similarchemically defined medium with a final volume of approximately 3.3 L or230 L respectively. The culture was initially grown in batch mode untilcarbon source depletion. After carbon source depletion a feed limitingthe carbon source was fed at an exponentially increasing rate. After theaddition of a defined quantity of carbon source the rate of feedsolution addition was decreased and IPTG was added to induce expressionof the Fab′. The fermentation was then continued and the Fab′accumulated in the periplasm. At a defined period after induction theculture was harvested by centrifugation and the Fab′ was extracted fromthe cells by resuspending the harvested cells in a Tris and EDTA bufferand heating to 59° C.

The growth profiles of the fermentations were determined by measuringthe optical density of culture at 600 nm.

The Fab′ titres were determined by Protein G HPLC as described inExample 5 above except that during the periplasmic extraction freshcells were used and 1 mL of extraction buffer was added to the cellculture. The supernatant and periplasmic Fab′ were measured as describedin Example 5. FIG. 8 shows the periplasmic Fab′ titre.

FIG. 7 shows the comparative growth profiles of 5 L and 200 Lfermentations of anti-TNF Fab′ and recombinant DsbC expressing E. colistrain MXE008.

FIG. 8 shows the comparative Fab′ titres of 5 L and 200 L fermentationsof anti-TNF Fab′ and recombinant DsbC expressing E. coli strain MXE008.

Example 8 Growth of E. Coli Strains Expressing Anti-TNF Fab′ and DsbCUsing Large Scale Fermentations

The following strains, as produced by example 4 were tested infermentation experiments comparing growth of the strain and theexpression of an anti-TNFα Fab′:

MXE008 expressing anti-TNF Fab′ and DsbC

MXE009 expressing anti-TNF Fab′ and DsbC

The fermentations were carried out as described above in Example 7 usinga 20 L production fermenter.

The growth profiles of the fermentations were determined by measuringthe optical density of culture at 600 nm.

The Fab′ titres were determined by Protein G HPLC as described inExample 7 above.

FIG. 9 shows the comparative growth profiles of fermentations ofanti-TNF Fab′ and recombinant DsbC expressing E. coli strains MXE008 andMXE009.

FIG. 10 shows the comparative periplasmic Fab′ titres of fermentationsof anti-TNF Fab′ and recombinant DsbC expressing E. coli strain MXE008and MXE009.

While this invention has been particularly shown and described withreference to preferred embodiments, it will be understood to thoseskilled in the art that various changes in form and detail may be madewithout departing from the scope of the invention as defined by theappendant claims.

The invention claimed is:
 1. A recombinant gram-negative bacterial cell,wherein the cell: a) comprises an exogenous recombinant polynucleotideencoding DsbC; and b) has reduced Tsp protein activity as compared to awild-type cell resulting from a mutation in the Tsp gene that encodes amutated Tsp protein having reduced protease activity or a mutation inthe Tsp gene or regulatory sequence of the Tsp gene that reduces oreliminates expression of the Tsp protein, and wherein the cell's genomeis isogenic to a wild-type bacterial cell except for: the mutation inthe Tsp gene, the presence of the recombinant polynucleotide encodingDsbC and optionally, one or more of: a) a mutant spr gene encoding amutant spr protein containing a mutation that is an amino acidsubstitution at one or more amino acids within the spr protein, whereinthe mutant spr protein reduces lysis of cells containing a mutated Tspgene; b) a mutated DegP gene encoding a DegP protein having chaperoneactivity and reduced protease activity; c) a mutated ptr gene, whereinthe mutated ptr gene encodes a Protease III protein having reducedprotease activity or is a knockout mutated ptr d) a mutated OmpT gene,wherein the mutated OmpT gene encodes a OmpT protein having reducedprotease activity or is a knockout mutated OmpT gene; and e) apolynucleotide sequence encoding a protein of interest.
 2. The cellaccording to claim 1, wherein the cell comprises the mutant spr geneencoding a mutant spr protein that reduces lysis of cells containing amutated Tsp gene.
 3. The cell according to claim 2, wherein the mutantspr gene encodes a mutant spr protein having a mutation at one or moreamino acids selected from the group consisting of H157, N31, R62, 170,Q73, C94, S95, V98, Q99, R100, L108, Y115, D133, V135, L136, G140, R144,H145, and G147, wherein the wild-type spr protein has the sequence ofSEQ ID NO:
 21. 4. The cell according to claim 3, wherein the mutant sprgene encodes a mutant spr protein having one or more mutations selectedfrom the group consisting of H157A, N31Y, R62C, 170T, Q73R, C94A, S95F,V98E, Q99P, R1000, L108S, Y115F, D133A, V135D, V1350, L136P, G140C,R144C, H145A, and G147C, wherein the wild-type spr protein has thesequence of SEQ ID NO:
 21. 5. The cell according to claim 4, wherein themutant spr gene encodes a mutant spr protein having one or moremutations selected from the group consisting of S95F, V98E, Y115F,D133A, V135D, V135G, and G147C, wherein the wild-type spr protein hasthe sequence of SEQ ID NO:
 21. 6. The cell according to claim 5, whereinthe mutant spr gene encodes a mutant spr protein having the mutationsS95F and Y115F, wherein the wild-type spr protein has the sequence ofSEQ ID NO:
 21. 7. The cell according to claim 4, wherein the mutant sprgene encodes a mutant spr protein having a mutation selected from thegroup consisting of C94A, D133A, H145A, and H157A, wherein the wild-typespr protein has the sequence of SEQ ID NO:
 21. 8. The cell according toclaim 1, wherein the cell comprises one or more of the following: a) themutated DegP gene encoding the DegP protein having chaperone activityand reduced protease activity; b) the mutated ptr gene, wherein themutated ptr gene encodes the Protease III protein having reducedprotease activity or is the knockout mutated ptr gene; and c) themutated OmpT gene, wherein the mutated OmpT gene encodes the OmpTprotein having reduced protease activity or is a knockout mutated OmpTgene.
 9. The cell according to claim 1, wherein the mutation in the Tspgene that eliminates expression of the Tsp protein comprises a knockoutmutation of the Tsp gene.
 10. The cell according to claim 9, wherein thecell's genome is isogenic to a wild-type bacterial cell from which thecell is derived except for the mutated Tsp gene, the recombinantpolynucleotide encoding DsbC and optionally, the mutated spr gene. 11.The cell according to claim 9, wherein the knockout mutation of the Tspgene comprises a mutation to the Tsp gene start codon and/or one or morestop codons positioned downstream of the gene start codon and upstreamof the Tsp gene stop codon.
 12. The cell according to claim 11, whereinthe knockout mutation of the Tsp gene comprises a restriction markersite created by a missense mutation to the Tsp gene start codon andoptionally, one or more additional point mutations.
 13. The cellaccording to claim 12, wherein the knockout mutated Tsp gene comprisesSEQ ID NO:3.
 14. The cell according to claim 1, wherein the cell is E.coli.
 15. The cell according to claim 1, wherein the cell comprises apolynucleotide sequence encoding a protein of interest.
 16. The cellaccording to claim 15, wherein the cell comprises a vector, said vectorcomprising both the recombinant polynucleotide encoding DsbC and thepolynucleotide encoding the protein of interest.
 17. The cell accordingto claim 16, wherein the vector comprises a promoter which controls theexpression of the recombinant polynucleotide encoding DsbC and thepolynucleotide sequence encoding a protein of interest.
 18. The cellaccording to claim 15, wherein the protein of interest is an antibody oran antigen binding fragment thereof.
 19. The cell according to claim 18,wherein the antibody or antigen binding fragment thereof is specific forTNF.
 20. A method for producing a protein of interest comprising: a)culturing a recombinant gram-negative bacterial cell as defined in claim1 that comprises a polynucleotide encoding a protein of interest in aculture medium under conditions effective to express the recombinantprotein of interest and the recombinant polynucleotide encoding DsbC;and b) recovering the protein of interest from the periplasm of therecombinant gram-negative bacterial cell and/or the culture medium. 21.The method according to claim 20, wherein the expression of thepolynucleotide sequence encoding a protein of interest and therecombinant polynucleotide encoding DsbC is induced by adding an inducerto the culture medium, wherein the polynucleotide encoding the proteinof interest and the polynucleotide encoding DsbC are under the controlof a promoter inducible by the inducer.
 22. The method according toclaim 20, wherein the method further comprises separating the protein ofinterest from DsbC.